MAGE peptides presented by HLA class II molecules

ABSTRACT

The invention describes HLA class II binding peptides encoded by the MAGE tumor associated genes, as well as nucleic acids encoding such peptides and antibodies relating thereto. The peptides stimulate the activity and proliferation of CD4 +  T lymphocytes. Methods and products also are provided for diagnosing and treating conditions characterized by expression of MAGE genes.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 09/860,840, filed May 18, 2001 now U.S. Pat. No. 7,049,413.

FIELD OF THE INVENTION

This invention relates to fragments of the tumor associated MAGEproteins which bind to and are presented to T lymphocytes by HLA classII molecules. The peptides, nucleic acid molecules which code for suchpeptides, as well as related antibodies and CD4⁺ T lymphocytes, areuseful, inter alia, in diagnostic and therapeutic contexts.

BACKGROUND OF THE INVENTION

The process by which the mammalian immune system recognizes and reactsto foreign or alien materials is complex. An important facet of thesystem is the T cell response, which in part comprises mature Tlymphocytes which are positive for either CD4 or CD8 cell surfaceproteins. T cells can recognize and interact with other cells via cellsurface complexes on the other cells of peptides and molecules referredto as human leukocyte antigens (“HLAs”) or major histocompatibilitycomplexes (“MHCs”). The peptides are derived from larger molecules whichare processed by the cells which also present the HLA/MHC molecule. SeeMale et al., Advanced Immunology (J.P. Lipincott Company, 1987),especially chapters 6-10. The interaction of T cells and complexes ofHLA/peptide is restricted, requiring a specific T cell for a specificcomplex of an HLA molecule and a peptide. If a specific T cell is notpresent, there is no T cell response even if its partner complex ispresent. Similarly, there is no response if the specific complex isabsent, but the T cell is present. The mechanisms described above areinvolved in the immune system's response to foreign materials, inautoimmune pathologies, and in responses to cellular abnormalities.

The T cell response to foreign antigens includes both cytolytic Tlymphocytes and helper T lymphocytes. CD8⁺ cytotoxic or cytolytic Tcells (CTLs) are T cells which, when activated, lyse cells that presentthe appropriate antigen presented by HLA class I molecules. CD4⁺ Thelper cells are T cells which secrete cytokines to stimulatemacrophages and antigen-producing B cells which present the appropriateantigen by HLA class II molecules on their surface.

The mechanism by which T cells recognize alien materials also has beenimplicated in cancer. A number of cytolytic T lymphocyte (CTL) clonesdirected against autologous melanoma have been described. In someinstances, the antigens recognized by these clones have beencharacterized. In De Plaen et al., Immunogenetics 40:360-369 (1994), the“MAGE” family, a family of genes encoding tumor specific antigens, isdescribed. (See also PCT application PCT/US92/04354, published on Nov.26, 1992.) The expression products of these genes are processed intopeptides which, in turn, are expressed on cell surfaces. This can leadto lysis of the tumor cells by specific CTLs. The genes are said to codefor “tumor rejection antigen precursors” or “TRAP” molecules, and thepeptides derived therefrom are referred to as “tumor rejection antigens”or “TRAs”. See Traversari et al., Immunogenetics 35: 145 (1992); van derBruggen et al., Science 254: 1643 (1991), for further information onthis family of genes. Also, see U.S. Pat. No. 5,342,774.

In U.S. Pat. No. 5,405,940, MAGE nonapeptides are taught which arepresented by the HLA-A1 molecule. Given the known specificity ofparticular peptides for particular HLA molecules, one should expect aparticular peptide to bind one HLA molecule, but not others. This isimportant, because different individuals possess different HLAphenotypes. As a result, while identification of a particular peptide asbeing a partner for a specific HLA molecule has diagnostic andtherapeutic ramifications, these are only relevant for individuals withthat particular HLA phenotype. There is a need for further work in thearea, because cellular abnormalities are not restricted to oneparticular HLA phenotype, and targeted therapy requires some knowledgeof the phenotype of the abnormal cells at issue.

In U.S. Pat. No. 5,591,430, additional isolated MAGE-A3 peptides aretaught which are presented by the HLA-A2 molecule. Therefore, a givenTRAP can yield a plurality of TRAs.

The foregoing references describe isolation and/or characterization oftumor rejection antigens which are presented by HLA class I molecules.These TRAs can induce activation and proliferation of CD8⁺ cytotoxic Tlymphocytes (CTLs) which recognize tumor cells that express the tumorassociated genes (e.g. MAGE genes) which encode the TRAs.

The importance of CD4⁺ T lymphocytes (helper T cells) in antitumorimmunity has been demonstrated in animal models in which these cells notonly serve cooperative and effector functions, but are also critical inmaintaining immune memory (reviewed by Topalian, Curr. Opin. Immunol.6:741-745, 1994). Moreover, several studies support the contention thatpoor tumor-specific immunity is due to inadequate activation of T helpercells.

It has recently been demonstrated that the tyrosinase gene encodespeptides which are presented by HLA class II molecules to stimulate CD4⁺T lymphocytes (Topalian et al., 1994; Yee et al., J. Immunol.157:4079-4086, 1996; Topalian et al., J. Exp. Med. 183:1965-1971, 1996).As with many cancer associated antigens, tyrosinase is expressed in alimited percentage of tumors and in limited types of tumors.Furthermore, the two identified MHC class II binding tyrosinase peptidesare HLA-DRB 1*0401-restricted peptides, recognized only by cells whichexpress the particular HLA molecule.

More recently, HLA class II peptide have been identified in the MAGE-A3,a cancer-testis antigen widely expressed in cancer cells but not innormal cells except testis. See U.S. Pat. No. 5,965,535 andPCT/US99/21230.

Although the cancer antigens tyrosinase and MAGE-A3 have been shown tocontain HLA class II binding peptides, there exist many patients whowould not benefit from any therapy which includes helper T cellstimulation via the aforementioned tyrosinase and MAGE-A3 peptides,either because the patient's tumor does not express tyrosinase, orbecause the patient does not express the appropriate HLA molecule.Accordingly, there is a need for the identification of additionalepitopes in tumor associated antigens that are presented by MHC class IImolecules and recognized by CD4⁺ lymphocytes.

SUMMARY OF THE INVENTION

It now has been discovered that the MAGE-A3 gene and other MAGE genesencode additional HLA class II binding peptides that are epitopespresented by HLA-DR1. These peptides, when presented by an antigenpresenting cell having the appropriate HLA class II molecule,effectively induce the activation and proliferation of CD4⁺ Tlymphocytes. In addition, T cell receptors that bind complexes of theMAGE HLA class II peptides and HLA class II molecules have been isolatedand sequenced.

The invention provides isolated MAGE-A3 peptides which bind HLA class IImolecules, and functional variants of such peptides, the functionalvariants comprising one or more amino acid additions, substitutions ordeletions to the MAGE-A3 peptide sequence. The invention also providesisolated nucleic acid molecules encoding such peptides, expressionvectors containing those nucleic acid molecules, host cells transfectedwith those nucleic acid molecules, and antibodies to those peptides andcomplexes of the peptides and HLA class II antigen presenting molecules.T lymphocytes which recognize complexes of the peptides and HLA class IIantigen presenting molecules are also provided. Kits and vaccinecompositions containing the foregoing molecules additionally areprovided. The foregoing can be used in the diagnosis or treatment ofconditions characterized by the expression of MAGE-A3, particularlycancer. As it is known that the members of the MAGE family ofpolypeptides and nucleic acids share significant sequence identity andfunctional homology (e.g., as tumor antigens and precursors), theinvention also embraces HLA binding peptides of similar amino acidsequence derived from members of the MAGE family other than MAGE-A3.Therefore, it is understood that the disclosure contained herein of MAGEHLA class II binding peptides, compositions containing such peptides,and methods of identifying and using such peptides applies also to othermembers of the MAGE tumor associated antigen family.

According to one aspect of the invention, isolated MAGE HLA classII-binding peptides are provided. The MAGE HLA class II binding peptidesinclude the amino acid sequence EFLWGPRA (SEQ ID NO:25). The MAGE HLAclass II binding peptides do not include a full length MAGE protein. Insome embodiments, the isolated peptides include or consist of an aminoacid sequence selected from the group consisting of SEQ ID NO:26, SEQ IDNO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ IDNO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ IDNO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ IDNO:42 and SEQ ID NO:63. In preferred embodiments, the peptides consistof an amino acid sequence selected from the group consisting of SEQ IDNO:26, SEQ ID NO:27, SEQ ID NO:28 and SEQ ID NO:29, particularly SEQ IDNO:26 or SEQ ID NO:27.

In certain embodiments, the isolated MAGE HLA class II-binding peptidesinclude an endosomal targeting signal. A preferred endosomal targetingsignal includes an endosomal targeting portion of human invariant chainIi.

In other embodiments, the isolated MAGE HLA class II-binding peptidesare non-hydrolyzable. Preferably the isolated peptides are selected fromthe group consisting of peptides comprising D-amino acids, peptidescomprising a -psi[CH₂NH]-reduced amide peptide bond, peptides comprisinga -psi[COCH₂]-ketomethylene peptide bond, peptides comprising a-psi[CH(CN)NH]-(cyanomethylene)amino peptide bond, peptides comprising a-psi[CH₂CH(OH)]-hydroxyethylene peptide bond, peptides comprising a-psi[CH₂O]-peptide bond, and peptides comprising a-psi[CH₂S]-thiomethylene peptide bond.

According to another aspect of the invention, compositions are providedthat include one or more isolated HLA class I-binding peptides and oneor more isolated MAGE HLA class II-binding peptides. In certainembodiments, the isolated MAGE HLA class II-binding peptide includes theamino acid sequence set forth as SEQ ID NO:25, but does not include afull length MAGE protein. In some embodiments, the HLA class I-bindingpeptides and the MAGE HLA class II-binding peptides are combined as apolytope polypeptide.

In preferred embodiments of the foregoing compositions, the one or moreisolated MAGE HLA class II-binding peptides include or consist of anamino acid sequence selected from the group consisting of SEQ ID NO:26,SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31,SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36,SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41,SEQ ID NO:42 and SEQ ID NO:63. In particularly preferred embodiments,the isolated MAGE HLA class II-binding peptides consist of an amino acidsequence selected from the group consisting of SEQ ID NO:26, SEQ IDNO:27, SEQ ID NO:28 and SEQ ID NO:29, preferably SEQ ID NO:26 or SEQ IDNO:27. The isolated MAGE HLA class II-binding peptides can include anendosomal targeting signal, preferably including an endosomal targetingportion of human invariant chain Ii.

According to still another aspect of the invention, compositions areprovided that include one or more of the foregoing isolated MAGE HLAclass II-binding peptides complexed with one or more isolated HLA classII molecules. In certain embodiments, the number of isolated MAGE HLAclass II-binding peptides and the number of isolated HLA class IImolecules are equal; preferably the isolated MAGE HLA class II-bindingpeptides and the isolated MAGE HLA class II molecules are coupled as atetrameric molecule of individual isolated MAGE HLA class II-bindingpeptides bound to individual isolated HLA class II molecules. Inpreferred embodiments, the HLA class II molecules are DR1 molecules.

In still other embodiments of these compositions, the MAGE HLA class IIbinding peptides and the HLA class II molecules are covalently linked.Preferably the covalently link between the MAGE HLA class II bindingpeptides and the HLA class II molecules includes a linker molecule.

According to yet another aspect of the invention, isolated nucleic acidmolecules are provided that encode the foregoing MAGE HLA class IIbinding peptides. The nucleic acid molecules do not encode a full lengthMAGE protein. In some embodiments, e nucleic acid molecule includes anucleotide sequence encoding a peptide selected from the groupconsisting of SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29,SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34,SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39,SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42 and SEQ ID NO:63.

In another aspect of the invention, expression vectors are provided thatinclude the foregoing isolated nucleic acids operably linked to apromoter. In some embodiments, the expression vectors also include anucleic acid which encodes an HLA-DR1 molecule. Host cell transfected ortransformed with the foregoing expression vectors also are provided. Insome embodiments, the host cell expresses an HLA-DR1 molecule.

According to a further aspect of the invention, methods for enrichingselectively a population of T lymphocytes with CD4⁺ T lymphocytesspecific for a MAGE HLA class II-binding peptide are provided. Themethods include contacting a population of T lymphocytes with an agentpresenting a complex of at least one of the foregoing MAGE HLA classII-binding peptide and an HLA class II molecule in an amount sufficientto selectively enrich the isolated population of T lymphocytes with theCD4⁺ T lymphocytes. In some embodiments, the HLA class II molecule is anHLA-DR1 molecule. In one preferred embodiment, the agent is a dendriticcell loaded with a MAGE HLA class II binding peptide that includes SEQID NO:25.

According to yet another aspect of the invention, methods for diagnosinga cancer characterized by expression of a MAGE protein are provided. Themethods include contacting a biological sample isolated from a subjectwith an agent that specifically binds at least one of the foregoing MAGEHLA class II binding peptides, and determining the binding between theagent and the MAGE HLA class II binding peptide as an indication of thecancer. In some embodiments, the agent is an antibody or an antigenbinding fragment thereof.

According to still another aspect of the invention, methods fordiagnosing a cancer characterized by expression of one of the foregoingMAGE HLA class II-binding peptides that forms a complex with an HLAclass II molecule are provided. The methods include contacting abiological sample isolated from a subject with an agent thatspecifically binds the complex; and determining binding between thecomplex and the agent as a determination of the disorder. Preferably theHLA class II molecule is an HLA-DR1 molecule. In preferred embodiments,the agent is a T lymphocyte, an antibody specific for the complex or amultimeric complex of T cell receptors.

According to another aspect of the invention, methods for treating asubject having a cancer characterized by expression of MAGE protein areprovided. The methods include administering to the subject an amount ofat least one of the foregoing MAGE HLA class II-binding peptideseffective to ameliorate the cancer.

In another aspect of the invention, methods for treating a subjecthaving a cancer characterized by expression of a MAGE protein areprovided. The methods include administering to the subject an amount ofat least one HLA class I-binding peptide and an amount of at least oneof the foregoing MAGE HLA class II-binding peptides effective toameliorate the cancer. In some embodiments, the HLA class I-bindingpeptides and the MAGE HLA class II-binding peptides are combined as apolytope polypeptide. Preferably, the HLA class I-binding peptide is aMAGE HLA class I-binding peptide.

According to a further aspect of the invention, methods for treating asubject having a cancer characterized by expression of a MAGE proteinare provided. The methods include administering to the subject an amountof an agent which enriches selectively in the subject the presence ofcomplexes of an HLA class II molecule and at least one of the foregoingMAGE HLA class II-binding peptides, sufficient to ameliorate the cancer.Preferably the the HLA class II molecule is a HLA-DR1 molecule. Incertain embodiments, the agent includes at least one of the foregoingMAGE HLA class II binding peptides. A preferred agent is a dendriticcell loaded with a MAGE HLA class II binding peptide that comprises SEQID NO:25.

According to still another aspect of the invention, methods for treatinga subject having a cancer characterized by expression of a MAGE proteinare provided. The methods include administering to the subject an amountof autologous CD4⁺ T lymphocytes sufficient to ameliorate the cancer.The CD4⁺ T lymphocytes are specific for complexes of an HLA class IImolecule and at least one of the foregoing MAGE HLA class II-bindingpeptides. Preferably, the HLA class II molecule is an HLA-DR1 molecule.

Also provided in accordance with an aspect of the invention are isolatedpolypeptides that bind selectively one of the foregoing polypeptides,provided that the isolated polypeptide is not an HLA class II molecule.Preferably the isolated polypeptide is an antibody. In some of theseembodiments the antibody is a monoclonal antibody, preferably a humanantibody, a humanized antibody, a chimeric antibody or a single chainantibody. In other embodiments the isolated polypeptide is an antibodyfragment selected from the group consisting of a Fab fragment, a F(ab)₂fragment, a Fv fragment or a fragment including a CDR3 region selectivefor a MAGE HLA class II-binding peptide.

According to a further aspect of the invention, isolated CD4⁺ Tlymphocytes that selectively bind a complex of an HLA class II moleculeand at least one of the foregoing MAGE HLA class II-binding peptides areprovided. Preferably the HLA class II molecule is an HLA-DR1 molecule.

According to still another aspect of the invention, isolated antigenpresenting cells are provided that include a complex of an HLA class IImolecule and at least one of the foregoing MAGE HLA class II-bindingpeptides. Preferably the HLA class II molecule is an HLA-DR1 molecule.

According to yet another aspect of the invention, methods for treating asubject having a cancer characterized by expression of a MAGE proteinare provided. The methods include administering to the subject an amountof CD4⁺ T lymphocytes sufficient to ameliorate the cancer, wherein theCD4⁺ T lymphocytes express at least one T cell receptor sequence thatincludes a CDR3 sequence selected from the group consisting of SEQ IDNO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, and SEQID NO:62. The T cell receptors preferably include an α chain and a βchain. In certain preferred embodiments, the α chain and β chain are apair of molecules including a pair of CDR3 sequences selected from thegroup consisting of (1) SEQ ID NO:47 and SEQ ID NO:50; (2) SEQ ID NO:53and SEQ ID NO:56; and (3) SEQ ID NO:59 and SEQ ID NO:62.

In some embodiments, the CD4⁺ T lymphocytes are autologous CD4⁺ Tlymphocytes that are transfected or transduced with a vector to expressthe at least one T cell receptor sequence. Preferably the vector is oneor more expression plasmids or viruses. A particularly preferred virusis a retrovirus.

In another aspect of the invention, methods for preparing a modified Tlymphocyte are provided. The methods include isolating peripheral bloodcells from a subject and transfecting or transducing the peripheralblood cells with one or more vectors that expresses at least one T cellreceptor sequence that includes a CDR3 sequence selected from the groupconsisting of SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56,SEQ ID NO:59, and SEQ ID NO:62. In certain embodiments the vector is anexpression plasmid or a virus, preferably a retrovinis.

In additional embodiments, the one or more vectors express an α chainand a β chain. Preferably the α chain and β chain are a pair ofmolecules including a pair of CDR3 sequences selected from the groupconsisting of (1) SEQ ID NO:47 and SEQ ID NO:50; (2) SEQ ID NO:53 andSEQ ID NO:56; and (3) SEQ ID NO:59 and SEQ ID NO:62.

According to another aspect of the invention, methods for treating asubject having a cancer characterized by expression of MAGE protein areprovided. The methods include isolating peripheral blood cells from asubject, transfecting or transducing the peripheral blood cells with oneor more vectors that expresses at least one T cell receptor sequencethat comprises a CDR3 sequence selected from the group consisting of SEQID NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, andSEQ ID NO:62, and administering the transfected or transduced cells tothe subject in an amount effective to ameliorate the cancer. In certainembodiments, the at least one T cell receptor sequence includes a pairof molecules that are a TCR α chain and a TCR β chain including a pairof CDR3 sequences selected from the group consisting of (1) SEQ ID NO:47and SEQ ID NO:50; (2) SEQ ID NO:53 and SEQ ID NO:56; and (3) SEQ IDNO:59 and SEQ ID NO:62.

According to still another aspect of the invention, isolated Tlymphocytes are provided that include at least one T cell receptorsequence that includes a CDR3 sequence selected from the groupconsisting of SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56,SEQ ID NO:59, and SEQ ID NO:62. Preferably the T lymphocytes include Tcell receptors including an α chain and a β chain. In some of theseembodiments, the α chain and β chain are a pair of molecules including apair of CDR3 sequences selected from the group consisting of (1) SEQ IDNO:47 and SEQ ID NO:50; (2) SEQ ID NO:53 and SEQ ID NO:56; and (3) SEQID NO:59 and SEQ ID NO:62.

The invention in another aspect provides synthetic multivalent T cellreceptor (TCR) complexes for binding to a MHC-peptide complex. The TCRcomplexes include a plurality of T cell receptor sequences including aCDR3 sequence selected from the group consisting of SEQ ID NO:47, SEQ IDNO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, and SEQ ID NO:62.Preferably the T cell receptors include an α chain and a β chain. Insome of these embodiments, the α chain and β chain are a pair ofmolecules including a pair of CDR3 sequences selected from the groupconsisting of (1) SEQ ID NO:47 and SEQ ID NO:50; (2) SEQ ID NO:53 andSEQ ID NO:56; and (3) SEQ ID NO:59 and SEQ ID NO:62.

In certain embodiments, the α chain and β chain are soluble forms of theT cell receptor α and β chains. In other embodiments, the T cellreceptors are in the form of multimers of two or more T cell receptors;a preferred multimer is a tetramer. In certain of these embodiments theT cell receptors are associated with one another via a linker molecule.Preferably the linker molecule is a multivalent attachment molecule.Particularly preferred multivalent attachment molecules include avidinand streptavidin, in which embodiments it is preferred that the T cellreceptors are biotinylated.

In still other embodiments, the foregoing TCR complexes also include adetectable label. Preferred detectable labels are fluorophores,chromophores, or radioactive molecules. In other embodiments, the TCRcomplexes include a therapeutic agent. Preferred therapeutic agentinclude cytotoxic agents, radioactive therapeutic agents andimmunostimulating agents. In additional embodiments, the TCR complexesare included in a pharmaceutically acceptable formulation.

According to another aspect of the invention, recombinant soluble T cellreceptors (TCR) are provided. The recombinant soluble T cell receptorsinclude a TCR α chain comprising a CDR3 region selected from the groupconsisting of SEQ ID NO:47, SEQ ID NO:53 and SEQ ID NO:59; and a TCR βchain comprising a CDR3 region selected from the group consisting of SEQID NO:50, SEQ ID NO:56 and SEQ ID NO:62. In some embodiments, the TCR isconstructed as a single chain TCR molecule. In other embodiments, the αchain and the β chain each include a dimerization moiety. Preferreddimerization moieties include immunoglobulin sequences and leuciriezippers.

The invention also provides pharmaceutical preparations containing anyone or more of the medicaments described above or throughout thespecification. Such pharmaceutical preparations can includepharmaceutically acceptable diluent carriers or excipients.

The use of the foregoing compositions, peptides and nucleic acids in thepreparation of a medicament, particularly a medicament for treatment ofcancer, also is provided.

These and other objects of the invention will be described in furtherdetail in connection with the detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows that a CD4⁺ T cell clone directed against a MAGE-A3 derivedantigen.

FIG. 2 is a graph showing titration of the MAGE-A3 peptide recognized byCD4+clone MAGJ569/F4.3. In FIG. 2A, the following peptides were tested:GSDPACYEFLWGPRAL (MAGE-A3₂₆₃₋₂₇₈; SEQ ID NO:3), ACYEFLWGPRALVETS(MAGE-A3₂₆₇₋₂₈₂; SEQ ID NO:4), ACYEFLWGPRALVE (SEQ ID NO:7),ACYEFLWGPRALV (SEQ ID NO:8), ACYEFLWGPRAL (SEQ ID NO:9), ACYEFLWGPRA(SEQ ID NO:10), ACYEFLWGPR (SEQ ID NO:11), CYEFLWGPRALVE (SEQ ID NO:12),YEFLWGPRALVE (SEQ ID NO:13) and EFLWGPRALVE (SEQ ID NO:14). In FIG. 2B,the following peptides were tested: GSDPACYEFLWGPRAL (SEQ ID NO:3),ACYEFLWGPRALVETS (SEQ ID NO:4), ACYEFLWGPRALVE (SEQ ID NO:7) andACYEFLWGPRALV (SEQ ID NO:8).

FIG. 3 is a graph showing that the MAGE-A3 peptide is presented to CD4⁺clone MAGJ569/F4.3 by HLA-DR molecules

FIG. 4 is a graph depicting recognition of DR1 tumor cells expressingMAGE-A3 by CD4⁺ clone MAGJ569/F4.3.

FIG. 5 shows recognition of a MAGE-3-derived antigen by three CD4⁺ Tcell clones.

FIG. 6 depicts the recognition of peptides by each of the three CD4⁺clones. The peptides tested were: GSDPACYEFLWGPRAL (SEQ ID NO:3),ACYEFLWGPRALVETS (SEQ ID NO:4), ACYEFLWGPRALVET (SEQ ID NO:16),ACYEFLWGPRALVE (SEQ ID NO:7), ACYEFLWGPRALV (SEQ ID NO:8), ACYEFLWGPRAL(SEQ ID NO:9), and ACYEFLWGPRA (SEQ ID NO:10).

FIG. 7 shows recognition of tumor cell lines by T cell clones. FIG. 7A:HLA-DR1 melanoma lines NA41-MEL and MZ2-MEL.43 express MAGE-3. HLA-DR1DDHK2-EBV B cells are autologous to the CD4⁺ T cells. Positive controlswere obtained by transduction of DDHK2-EBV with a retroviral constructencoding a truncated human invariant chain (Ii) fused with the MAGE-3protein (retro-Ii.MAGE-3). Cells were distributed in flat-bottomedmicrowells (2×10⁴ cells per well) and, if indicated, pulsed for 2 h with5 μg/ml of peptide ACYEFLWGPRALVETS (SEQ ID NO:4) and washed. 5,000 CD4⁺T cells were added to the stimulator cells. IFN-γ production wasmeasured by ELISA after 20 h of co-culture. The results shown representthe average and standard deviation of triplicate co-cultures. FIG. 7B:Chromium-labeled target cells were tested for lysis by clone 1.

FIG. 8 depicts titration of the MAGE-3 protein. HLA-DR1 dendritic cells(2×10⁴ cells per flat-bottomed microwell) were cultured for 24 h withdifferent concentrations of the MAGE-3^(bacteria) protein, washed, andincubated with 5×10³ cells per well from each CD4⁺ T cell clone. IFN-γproduction was measured by ELISA after 20 h of co-culture.

FIG. 9 shows the recognition of different MAGE peptides by the threeCD4⁺ T cell clones. FIG. 9A: DDHK2-EBV B cells (2×10⁴) were incubatedfor 2 h with the indicated peptides. 5×10³ cells from each autologousCD4⁺ T cell clone were added and IFN-γ production in the supernatant wasmeasured by ELISA after overnight co-culture. FIG. 9B:LB2095-EBV.retro-MAGE-1 and LB2348-EBV.retro-MAGE-4 were lysed byfreeze-thawing. HLA-DR1 dendritic cells (2.5×10⁴ cells per well) werecultured for 24 h with lysates at the equivalent of 5×10⁴ cells per wellof LB2095-EBV transfected with retro-MAGE-A1 and LB2348-EBV transfectedwith retro-MAGE-4. After washing, they were incubated with 5×10³ cellsper well from clone 1. IFN-γ production was measured after 20 h byELISA. The results shown represent the average of triplicateco-cultures. The peptides tested were: ACYEFLWGPRALVETS (SEQ ID NO:4),ACIEFLWGPRALIETS (SEQ ID NO:26), ACYEFLWGPRALIETS (SEQ ID NO:27),ACYEFLWGPRAHAETS (SEQ ID NO:29), ARYEFLWGPRALAETS (SEQ ID NO:30) andARYEFLWGPRAHAEIR (SEQ ID NO:31).

DETAILED DESCRIPTION OF THE INVENTION

The invention provides isolated MAGE peptides presented by HLA class IImolecules, which peptides stimulate the proliferation and activation ofCD4⁺ T lymphocytes. Such peptides are referred to herein as “MAGE HLAclass II binding peptides,” “HLA class II binding peptides,” “MHC classII binding peptides,” “MAGE epitopes” and the like. Hence, one aspect ofthe invention is an isolated peptide which includes the amino acidsequence of SEQ ID NO:25. The peptides referred to herein as “MAGE HLAclass II binding peptides” include fragments of MAGE proteins, but donot include any of the full-length MAGE proteins. Likewise, nucleicacids that encode the “MAGE HLA class II binding peptides” includefragments of the MAGE gene coding regions, but do not include thefull-length MAGE coding regions. As used herein, “MAGE” or “MAGE family”genes or proteins include the sequences known in the art as expressedpreferentially in cancer and testis tissues, including MAGE-A1, MAGE-A2,MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10,MAGE-A11, MAGE-A12, MAGE-B1, MAGE-B2, MAGE-B3, MAGE-C1, MAGE-C2, etc.The MAGE HLA class II binding peptides exclude, in preferredembodiments, certain peptides that previously were known, such as SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10.,SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:16.

The examples below show the isolation of peptides which are MAGE HLAclass II binding peptides. Some of these exemplary peptides areprocessed translation products of the MAGE-A3 nucleic acid (SEQ ID NO:1,the polypeptide sequence of which is given as SEQ ID NO:2), and some ofthe peptides are processed translation products of other MAGE nucleicacids. As such, it will be appreciated by one of ordinary skill in theart that the translation products from which a MAGE HLA class II bindingpeptide is processed to a final form for presentation may be of anylength or sequence so long as they encompass the MAGE HLA class IIbinding peptide. As demonstrated in the examples below, peptides orproteins as small as 13 or 14 amino acids and as large as the amino acidsequence of the MAGE-A3 protein (SEQ ID NO:2) are appropriatelyprocessed, presented by HLA class II molecules and effective instimulating CD4⁺ T lymphocytes. MAGE HLA class II binding peptides, suchas the peptides of SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ IDNO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ IDNO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ IDNO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42 and SEQ ID NO:63 mayhave one, two, three, four, five, six, seven, eight, nine, ten, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more amino acidsadded to either or both ends. The antigenic portion of such a peptide iscleaved out under physiological conditions for presentation by HLA classII molecules. It is also well known in the art that HLA class II peptidelength is variable between about 10 amino acids and about 30 amino acids(Engelhard, Ann. Rev. Immunol. 12:181-201, 1994). Most of the HLA classII binding peptides fall in to the length range of 12-19 amino acids.Nested sets of HLA class II binding peptides have been identified,wherein the peptides share a core sequence but have different aminoacids at amino and/or carboxyl terminal ends (see, e.g., Chicz et al.,J. Exp. Med. 178:27-47, 1993). Thus additional MAGE HLA class II bindingpeptides, as well as MAGE family HLA class II binding peptides, can beidentified by one of ordinary skill in the art according to theprocedures described herein.

The procedures described in the Examples can be utilized to identifyMAGE family HLA class II binding peptides. Thus, for example, one canload antigen presenting cells, such as dendritic cells of normal blooddonors, with a recombinant MAGE protein (or a fragment thereof) bycontacting the cells with the MAGE polypeptide or by introducing intothe cells a nucleic acid molecule which directs the expression of theMAGE protein of interest (or fragment thereof containing the HLA classII binding peptide). The antigen-presenting cells then can be used toinduce in vitro the activation and proliferation of specific CD4lymphocytes which recognize MAGE HLA class II binding peptides. Thesequence of the peptides then can be determined as described in theExamples, e.g., by stimulating cells with peptide fragments of the MAGEprotein used to stimulate the activation and proliferation of CD4lymphocytes. Alternatively, one can load antigen presenting cells withpeptides derived from a MAGE protein. For example, one can makepredictions of peptide sequences derived from MAGE family proteins whichare candidate HLA class II binding peptides based on the similarity withthe peptide sequences identified herein, and/or based on the consensusamino acid sequences for binding HLA class II molecules. In this regard,see, e.g. International applications PCT/US96/03182 and PCT/US98/01373.Peptides which are thus selected can be used in the assays describedherein for inducing specific CD4 lymphocytes and identification ofpeptides. Additional methods of selecting and testing peptides for HLAclass II binding are well known in the art.

As noted above, the invention embraces functional variants of MAGE HLAclass II binding peptides. As used herein, a “functional variant” or“variant” of a HLA class II binding peptide is a peptide which containsone or more modifications (generally 5 or fewer) to the primary aminoacid sequence of a HLA class II binding peptide and retains the HLAclass II and T cell receptor binding properties disclosed herein.Modifications which create a MAGE HLA class II binding peptidefunctional variant can be made for example 1) to enhance a property of aMAGE HLA class II binding peptide, such as peptide stability in anexpression system or the stability of protein-protein binding such asHLA-peptide binding; 2) to provide a novel activity or property to aMAGE HLA class II binding peptide, such as addition of an antigenicepitope or addition of a detectable moiety; or 3) to provide a differentamino acid sequence that produces the same or similar T cell stimulatoryproperties. Modifications to MAGE HLA class II binding peptides can bemade to nucleic acids which encode the peptides, and can includedeletions, point mutations, truncations, amino acid substitutions andadditions of amino acids. Alternatively, modifications can be madedirectly to the polypeptide, such as by cleavage, addition of a linkermolecule, addition of a detectable moiety, such as biotin, addition of afatty acid, substitution of one amino acid for another and the like.Variants also can be selected from libraries of peptides, which can berandom peptides or peptides based on the sequence of the MAGE peptidesincluding substitutions at one or more positions. Some suitablesequences for preparation of peptide libraries are provided in theExamples below. For example, a peptide library can be used incompetition assays with complexes of MAGE peptides bound to HLA class IImolecules (e.g. dendritic cells loaded with MAGE peptide). Peptideswhich compete for binding of the MAGE peptide to the HLA class IImolecule can be sequenced and used in other assays (e.g., CD4 lymphocyteproliferation) to determine suitability as MAGE peptide functionalvariants.

Modifications also embrace fusion proteins comprising all or part of aMAGE HLA class II binding peptide amino acid sequence, such as theinvariant chain-MAGE-A3 fusion proteins described herein. The inventionthus embraces fusion proteins comprising MAGE HLA class II bindingpeptides and endosomal targeting signals such as the human invariantchain (Ii). As is disclosed below, fusion of an endosomal targetingportion of the human invariant chain to MAGE-A3 resulted in efficienttargeting of MAGE-A3 to the HLA class II peptide presentation pathway.An “endosomal targeting portion” of the human invariant chain or othertargeting polypeptide is that portion of the molecule which, when fusedor conjugated to a second polypeptide, increases endosomal localizationof the second polypeptide. Thus endosomal targeting portions can includethe entire sequence or only a small portion of a targeting polypeptidesuch as human invariant chain Ii. One of ordinary skill in the art canreadily determine an endosomal targeting portion of a targetingmolecule.

Prior investigations (e.g., PCT/US99/21230) noted that fusion of anendosomal targeting portion of LAMP-1 protein did not significantlyincrease targeting of MAGE-A3 to the HLA class II peptide presentationpathway. Therefore, the particular MAGE peptides of the invention can betested as fusions with LAMP-I to determine if such fusion proteins areefficiently targeted to the HLA class II peptide presentation pathway.Additional endosomal targeting signals can be identified by one ofordinary skill in the art, fused to MAGE-A3 or a MAGE-A3 HLA class IIbinding portion thereof, or other MAGE HLA class II binding peptides,and tested for targeting to the HLA class II peptide presentationpathway using no more than routine experimentation.

The amino acid sequence of MAGE HLA class II binding peptides may be ofnatural or non-natural origin, that is, they may comprise a natural MAGEHLA class II binding peptide molecule or may comprise a modifiedsequence as long as the amino acid sequence retains the ability tostimulate helper T cells when presented (i.e., bound to an appropriateHLA class II molecule on the cell surface) and retains the property ofbinding to an HLA class II molecule such as an HLA DR1 molecule. Suchmodified peptides that retain the ability to bind HLA class II moleculesand stimulate helper T cells are “functional variants” as used herein.For example, MAGE HLA class II binding peptides in this context may befusion proteins including a MAGE HLA class II binding peptide andunrelated amino acid sequences, synthetic MAGE HLA class II bindingpeptides, labeled peptides, peptides isolated from patients with a MAGEprotein-expressing cancer, peptides isolated from cultured cells whichexpress one or more MAGE proteins, peptides coupled to nonpeptidemolecules (for example in certain drug delivery systems) and othermolecules which include the amino acid sequence of SEQ ID NO:25,preferably including an amino acid sequence selected from SEQ ID NO:26,SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31,SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36,SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41,SEQ ID NO:42 and SEQ ID NO:63.

Preferably, the MAGE HLA class II binding peptides are non-hydrolyzable.To provide such peptides, one may select MAGE HLA class II bindingpeptides from a library of non-hydrolyzable peptides, such as peptidescontaining one or more D-amino acids or peptides containing one or morenon-hydrolyzable peptide bonds linking amino acids. Alternatively, onecan select peptides which are optimal for inducing CD4⁺ T lymphocytesand then modify such peptides as necessary to reduce the potential forhydrolysis by proteases. For example, to determine the susceptibility toproteolytic cleavage, peptides may be labeled and incubated with cellextracts or purified proteases and then isolated to determine whichpeptide bonds are susceptible to proteolysis, e.g., by sequencingpeptides and proteolytic fragments. Alternatively, potentiallysusceptible peptide bonds can be identified by comparing the amino acidsequence of a MAGE HLA class II binding peptide with the known cleavagesite specificity of a panel of proteases. Based on the results of suchassays, individual peptide bonds which are susceptible to proteolysiscan be replaced with non-hydrolyzable peptide bonds by in vitrosynthesis of the peptide.

Many non-hydrolyzable peptide bonds are known in the art, along withprocedures for synthesis of peptides containing such bonds.Non-hydrolyzable bonds include, but are not limited to,-psi[CH₂NH]-reduced amide peptide bonds, -psi[COCH₂]-ketomethylenepeptide bonds, -psi [CH(CN)NH]-(cyanomethylene)amino peptide bonds, -psi[CH₂CH(OH)]-hydroxyethylene peptide bonds, -psi[CH₂O]-peptide bonds, and-psi[CH₂S]thiomethylene peptide bonds.

Nonpeptide analogs of peptides, e.g., those which provide a stabilizedstructure or lessened biodegradation, are also provided in accordancewith the invention. Peptide mimetic analogs can be prepared based on aselected MAGE HLA class II binding peptide by replacement of one or moreresidues by nonpeptide moieties. Preferably, the nonpeptide moietiespermit the peptide to retain its natural conformation, or stabilize apreferred, e.g., bioactive, confirmation. Such peptides can be tested inmolecular or cell-based binding assays to assess the effect of thesubstitution(s) on conformation and/or activity. One example of methodsfor preparation of nonpeptide mimetic analogs from peptides is describedin Nachman et al., Regul. Pept. 57:359-370 (1995). Peptide as usedherein embraces all of the foregoing.

If a variant involves a change to an amino acid sequence of theinvention, such as SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ IDNO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ IDNO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ IDNO:39, SEQ ID NO:40, SEQ ID NO:41 and SEQ ID NO:42, functional variantsof the MAGE HLA class II binding peptide having conservative amino acidsubstitutions typically will be preferred, i.e., substitutions whichretain a property of the original amino acid such as charge,hydrophobicity, conformation, etc. Examples of conservativesubstitutions of amino acids include substitutions made amongst aminoacids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K,R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

Other methods for identifying functional variants of the MAGE HLA classII binding peptides are provided in a published PCT application ofStrominger and Wucherpfennig (PCT/US96/03182). These methods rely uponthe development of amino acid sequence motifs to which potentialepitopes may be compared. Each motif describes a finite set of aminoacid sequences in which the residues at each (relative) position may be(a) restricted to a single residue, (b) allowed to vary amongst arestricted set of residues, or (c) allowed to vary amongst all possibleresidues. For example, a motif might specify that the residue at a firstposition may be any one of the residues valine, leucine, isoleucine,methionine, or phenylalanine; that the residue at the second positionmust be histidine; that the residue at the third position may be anyamino acid residue; that the residue at the fourth position may be anyone of the residues valine, leucine, isoleucine, methionine,phenylalanine, tyrosine or tryptophan; and that the residue at the fifthposition must be lysine.

Other computational methods for selecting amino acid substitutions, suchas iterative computer structural modeling, can also be performed by oneof ordinary skill in the art to prepare variants. Sequence motifs forMAGE HLA class II binding peptide functional variants can be developedby analysis of the binding domains or binding pockets of majorhistocompatibility complex HLA-DR proteins and/or the T cell receptor(“TCR”) contact points of the MAGE HLA class II binding peptidesdisclosed herein. By providing a detailed structural analysis of theresidues involved in forming the HLA class II binding pockets, one isenabled to make predictions of sequence motifs for binding of MAGEpeptides to any of the HLA class II proteins.

Using these sequence motifs as search, evaluation, or design criteria,one is enabled to identify classes of peptides (e.g. MAGE HLA class IIbinding peptides, particularly the MAGE peptides disclosed herein, andfunctional variants thereof) which have a reasonable likelihood ofbinding to a particular HLA molecule and of interacting with a T cellreceptor to induce T cell response. These peptides can be synthesizedand tested for activity as described herein. Use of these motifs, asopposed to pure sequence homology (which excludes many peptides whichare antigenically similar but quite distinct in sequence) or sequencehomology with unlimited “conservative” substitutions (which admits manypeptides which differ at critical highly conserved sites), represents amethod by which one of ordinary skill in the art can evaluate peptidesfor potential application in the treatment of disease.

The Strominger and Wucherpfennig PCT application, and references citedtherein, all of which are incorporated by reference, describe the HLAclass II and TCR binding pockets which contact residues of an HLA classII peptide. By keeping the residues which are likely to bind in the HLAclass II and/or TCR binding pockets constant or permitting onlyspecified substitutions, functional variants of MAGE HLA class IIbinding peptides can be prepared which retain binding to HLA class IIand T cell receptor.

Thus methods for identifying additional MAGE family HLA class IIpeptides, in particular MAGE HLA class II binding peptides, andfunctional variants thereof, are provided. In general, any MAGE proteincan be subjected to the analysis noted above, peptide sequences selectedand the tested as described herein. With respect to MAGE proteinsdisclosed herein, for example, the methods include selecting a MAGE HLAclass II binding peptide, an HLA class II binding molecule which bindsthe MAGE HLA class II binding peptide, and a T cell which is stimulatedby the MAGE HLA class II binding peptide presented by the HLA class IIbinding molecule. In preferred embodiments, the MAGE HLA class IIbinding peptide comprises the amino acid sequence of SEQ ID NO:25, moreparticularly an amino acid sequence selected from SEQ ID NO:26, SEQ IDNO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ IDNO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ IDNO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41 and SEQ IDNO:42. More preferably, the peptide consists of those amino acidsequences. A first amino acid residue of the MAGE HLA class II bindingpeptide is mutated to prepare a variant peptide. The amino acid residuecan be mutated according to the principles of HLA and T cell receptorcontact points set forth in the Strominger and Wucherpfennig PCTapplication described above. Any method for preparing variant peptidescan be employed, such as synthesis of the variant peptide, recombinantlyproducing the variant peptide using a mutated nucleic acid molecule, andthe like.

The binding of the variant peptide to HLA class II binding molecule andstimulation of the T cell are then determined according to standardprocedures. For example, as exemplified below, the variant peptide canbe contacted with an antigen presenting cell which contains the HLAclass II molecule which binds the MAGE peptide to form a complex of thevariant peptide and antigen presenting cell. This complex can then becontacted with a T cell which recognizes the MAGE HLA class II bindingpeptide presented by the HLA class II binding molecule. T cells can beobtained from a patient having a condition characterized by expressionof MAGE proteins or nucleic acids, such as cancer. Recognition ofvariant peptides by the T cells can be determined by measuring anindicator of T cell stimulation such as TNF or IFNγ production. Similarprocedures can be carried out for identification and characterization ofother MAGE family HLA class II binding peptides. T cells, and othercells that have similar binding properties, also can be made using thecloned T cell receptors described herein, in accordance with standardtransfection or transduction procedures.

Binding of a variant peptide to the HLA class II binding molecule andstimulation of the T cell by the variant peptide presented by the HLAclass II binding molecule indicates that the variant peptide is afunctional variant. The methods also can include the step of comparingthe stimulation of the T cell by the MAGE HLA class II binding peptideand the stimulation of the T cell by the functional variant as adetermination of the effectiveness of the stimulation of the T cell bythe functional variant. By comparing the functional variant with theMAGE HLA class II binding peptide, peptides with increased T cellstimulatory properties can be prepared.

The foregoing methods can be repeated sequentially with, for example, asecond, third, fourth, fifth, sixth, seventh, eighth, ninth, and tenthsubstitutions to prepare additional functional variants of the disclosedMAGE HLA class II binding peptides.

Variants of the MAGE HLA class II binding peptides prepared by any ofthe foregoing methods can be sequenced, if necessary, to determine theamino acid sequence and thus deduce the nucleotide sequence whichencodes such variants.

Also a part of the invention are those nucleic acid sequences which codefor a MAGE HLA class II binding peptides or variants thereof and othernucleic acid sequences which hybridize to a nucleic acid moleculeconsisting of the above described nucleotide sequences, under highstringency conditions. Preferred nucleic acid molecules include thosecomprising the nucleotide sequences that encode SEQ ID NO:26, SEQ IDNO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ IDNO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ IDNO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ IDNO:42 and SEQ ID NO:63, which are portions of known MAGE gene codingregions (as described below). The term “high stringency hybridizationconditions” as used herein refers to parameters with which the art isfamiliar. Nucleic acid hybridization parameters may be found inreferences which compile such methods, e.g. Molecular Cloning: ALaboratory Manual, J. Sambrook, et al., eds., Second Edition, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, orCurrent Protocols in Molecular Biology, F. M. Ausubel, et al., eds.,John Wiley & Sons, Inc., New York. More specifically, high stringencyhybridization conditions, as used herein, refers to hybridization at 65°C. in hybridization buffer (3.5×SSC, 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin, 25 mM NaH₂PO₄ (pH 7), 0.5% SDS,2 mM EDTA). SSC is 0.15M sodium chloride/0.015M sodium citrate, pH 7;SDS is sodium dodecyl sulphate; and EDTA is ethylene diaminetetraaceticacid. After hybridization, the membrane upon which the DNA istransferred is washed at 2×SSC at room temperature and then at0.1-0.5×SSC/0.1×SDS at temperatures up to 68° C. Alternatively, highstringency hybridization may be performed using a commercially availablehybridization buffer, such as ExpressHyb™ buffer (Clontech) usinghybridization and washing conditions described by the manufacturer.

There are other conditions, reagents, and so forth which can used, whichresult in a similar degree of stringency. The skilled artisan will befamiliar with such conditions, and thus they are not given here. It willbe understood, however, that the skilled artisan will be able tomanipulate the conditions in a manner to permit the clear identificationof homologs and alleles of nucleic acids encoding the MAGE HLA class IIbinding peptides of the invention. The skilled artisan also is familiarwith the methodology for screening cells and libraries for expression ofsuch molecules which then are routinely isolated, followed by isolationof the pertinent nucleic acid molecule and sequencing.

In general homologs and alleles typically will share at least 90% aminoacid identity and/or at least 90% nucleotide identity to the amino acidsequence of a MAGE HLA class II binding peptide as described herein, ornucleic acids which encode such a peptide, respectively. In someinstances homologs and alleles will share at least 92% nucleotideidentity and/or at least 95% amino acid identity and in still otherinstances will share at least 95% nucleotide identity and/or at least99% amino acid identity. Still more preferably, homologs and alleleswill share at least 99% nucleotide identity and/or at least 99% aminoacid identity Complements of the foregoing nucleic acids also areembraced by the invention.

In screening for nucleic acids which encode a MAGE HLA class II bindingpeptide, a nucleic acid hybridization such as a Southern blot or aNorthern blot may be performed using the foregoing conditions, togetherwith a detectably labeled probe (e.g., radioactive such as ³²P,chemiluminescent, fluorescent labels). After washing the membrane towhich DNA encoding a MAGE HLA class II binding peptide was finallytransferred, the membrane can be placed against X-ray film,phosphorimager or other detection device to detect the detectable label.

The invention also includes the use of nucleic acid sequences whichinclude alternative codons that encode the same amino acid residues ofthe MAGE HLA class II binding peptides. For example, as disclosedherein, the peptide ACIEFLWGPRALIETS (SEQ ID NO:26) is a MAGE HLA classII binding peptide. The leucine residues (amino acids No. 6 and 12 ofSEQ ID NO:26) can be encoded by the codons CUA, CUC, CUG, CUU, UUA andUUG. Each of the six codons is equivalent for the purposes of encoding aleucine residue. Thus, it will be apparent to one of ordinary skill inthe art that any of the leucine-encoding nucleotide triplets may beemployed to direct the protein synthesis apparatus, in vitro or in vivo,to incorporate a leucine residue. Similarly, nucleotide sequencetriplets which encode other amino acid residues comprising the MAGE HLAclass II binding peptide of SEQ ID NO:26 include: CGA, CGC, CGG, CGT,AGA and AGG (arginine codons); GAA and GAG (glutamine codons); and UUCand UUU (phenylalanine codons). Other amino acid residues may be encodedsimilarly by multiple nucleotide sequences. Thus, the invention embracesdegenerate nucleic acids that differ from the native MAGE HLA class IIbinding peptide encoding nucleic acids in codon sequence due to thedegeneracy of the genetic code.

It will also be understood that the invention embraces the use of thesequences in expression vectors, as well as to transfect host cells andcell lines, be these prokaryotic (e.g., E. coli), or eukaryotic (e.g.,dendritic cells, CHO cells, COS cells, yeast expression systems andrecombinant baculovirus expression in insect cells). The expressionvectors require that the pertinent sequence, i.e., nucleotide sequencesthat encode the peptides described herein, be operably linked to apromoter. As it has been found that human HLA-DR1 molecules present aMAGE HLA class II binding peptide, the expression vector may alsoinclude a nucleic acid sequence coding for an HLA-DR1 molecule. In asituation where the vector contains both coding sequences, it can beused to transfect a cell which does not normally express either one. TheMAGE HLA class II binding peptide coding sequence may be used alone,when, e.g. the host cell already expresses an HLA-DR1 molecule. Ofcourse, there is no limit on the particular host cell which can be usedas the vectors which contain the two coding sequences may be used inhost cells which do not express HLA-DR1 molecules if desired, and thenucleic acid coding for the MAGE HLA class II binding peptide can beused in antigen presenting cells which express an HLA-DR1 molecule.

As used herein, “an HLA-DR1 molecule” includes the subtypes DRB1*010101,DRB1*010102, DRB1*010201, DRB1*010202, DRB*10103, DRB1*0104, DRB1*0105,DRB1*0106, DRB1*0107, DRB1*0108, DRB1*0109, DRB1*0110 and other subtypesknown to one of ordinary skill in the art. Other subtypes can be foundin various publications that include current HLA allele lists, such asthe websites of IMGT, the international ImMunoGeneTics informationsystem®, or EMBL-EBI (European Bioinformatics Institute).

As used herein, a “vector” may be any of a number of nucleic acids intowhich a desired sequence may be inserted by restriction and ligation fortransport between different genetic environments or for expression in ahost cell. Vectors are typically composed of DNA although RNA vectorsare also available. Vectors include, but are not limited to, plasmids,phagemids and virus genomes. A cloning vector is one which is able toreplicate autonomously or after integration into the genome in a hostcell, and which is further characterized by one or more endonucleaserestriction sites at which the vector may be cut in a determinablefashion and into which a desired DNA sequence may be ligated such thatthe new recombinant vector retains its ability to replicate in the hostcell. In the case of plasmids, replication of the desired sequence mayoccur many times as the plasmid increases in copy number within the hostbacterium or just a single time per host before the host reproduces bymitosis. In the case of phage, replication may occur actively during alytic phase or passively during a lysogenic phase. An expression vectoris one into which a desired DNA sequence may be inserted by restrictionand ligation such that it is operably joined to regulatory sequences andmay be expressed as an RNA transcript. Vectors may further contain oneor more marker sequences suitable for use in the identification of cellswhich have or have not been transformed or transfected with the vector.Markers include, for example, genes encoding proteins which increase ordecrease either resistance or sensitivity to antibiotics or othercompounds, genes which encode enzymes whose activities are detectable bystandard assays known in the art (e.g., β-galactosidase, luciferase oralkaline phosphatase), and genes which visibly affect the phenotype oftransformed or transfected cells, hosts, colonies or plaques (e.g.,green fluorescent protein). Preferred vectors are those capable ofautonomous replication and expression of the structural gene productspresent in the DNA segments to which they are operably joined.

Preferably the expression vectors contain sequences which target a MAGEfamily polypeptide, or a HLA class II binding peptide derived therefrom,to the endosomes of a cell in which the protein or peptide is expressed.HLA class II molecules contain an invariant chain (Ii) which impedesbinding of other molecules to the HLA class II molecules. This invariantchain is cleaved in endosomes, thereby permitting binding of peptides byHLA class II molecules. Therefore it is preferable that the MAGE HLAclass II binding peptides and precursors thereof (e.g. the various MAGEproteins that contain HLA class II binding peptides as identifiedherein) are targeted to the endosome, thereby enhancing MAGE HLA classII binding peptide binding to HLA class II molecules. Targeting signalsfor directing molecules to endosomes are known in the art and thesesignals conveniently can be incorporated in expression vectors such thatfusion proteins which contain the endosomal targeting signal areproduced. Sanderson et al. (Proc. Nat'l. Acad. Sci. USA 92:7217-7221,1995), Wu et al. (Proc. Nat'l. Acad. Sci. USA 92:11671-11675, 1995) andThomson et al (J. Virol. 72:2246-2252, 1998) describe endosomaltargeting signals (including invariant chain Ii and lysosomal-associatedmembrane protein LAMP-1) and their use in directing antigens toendosomal and/or lysosomal cellular compartments. As disclosed in theExamples, invariant chain-MAGE fusion proteins are preferred.

Endosomal targeting signals such as invariant chain also can beconjugated to MAGE proteins or peptides by non-peptide bonds (i.e. notfusion proteins) to prepare a conjugate capable of specificallytargeting MAGE proteins. Specific examples of covalent bonds includethose wherein bifunctional cross-linker molecules are used. Thecross-linker molecules may be homobifunctional or heterobifunctional,depending upon the nature of the molecules to be conjugated.Homobifunctional cross-linkers have two identical reactive groups.Heterobifunctional cross-linkers are defined as having two differentreactive groups that allow for sequential conjugation reaction. Varioustypes of commercially available cross-linkers are reactive with one ormore of the following groups; primary amines, secondary amines,sulfhydryls, carboxyls, carbonyls and carbohydrates. One of ordinaryskill in the art will be able to ascertain without undue experimentationthe preferred molecule for linking the endosomal targeting moiety andMAGE peptide or protein, based on the chemical properties of themolecules being linked and the preferred characteristics of the bond orbonds.

As used herein, a coding sequence and regulatory sequences are said tobe “operably” joined when they are covalently linked in such a way as toplace the expression or transcription of the coding sequence under theinfluence or control of the regulatory sequences. If it is desired thatthe coding sequences be translated into a functional protein, two DNAsequences are said to be operably joined if induction of a promoter inthe 5′ regulatory sequences results in the transcription of the codingsequence and if the nature of the linkage between the two DNA sequencesdoes not (1) result in the introduction of a frame-shift mutation, (2)interfere with the ability of the promoter region to direct thetranscription of the coding sequences, or (3) interfere with the abilityof the corresponding RNA transcript to be translated into a protein.Thus, a promoter region would be operably joined to a coding sequence ifthe promoter region were capable of effecting transcription of that DNAsequence such that the resulting transcript might be translated into thedesired protein or polypeptide.

The precise nature of the regulatory sequences needed for geneexpression may vary between species or cell types, but shall in generalinclude, as necessary, 5′ non-transcribed and 5′ non-translatedsequences involved with the initiation of transcription and translationrespectively, such as a TATA box, capping sequence, CAAT sequence, andthe like. In particular, such 5′ non-transcribed regulatory sequenceswill include a promoter region which includes a promoter sequence fortranscriptional control of the operably joined gene. Regulatorysequences may also include enhancer sequences or upstream activatorsequences as desired. The vectors of the invention may optionallyinclude 5′ leader or signal sequences. The choice and design of anappropriate vector is within the ability and discretion of one ofordinary skill in the art.

Expression vectors containing all the necessary elements for expressionare commercially available and known to those skilled in the art. See,e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, SecondEdition, Cold Spring Harbor Laboratory Press, 1989. Cells aregenetically engineered by the introduction into the cells ofheterologous DNA (RNA) encoding a MAGE HLA class II binding peptide.That heterologous DNA (RNA) is placed under operable control oftranscriptional elements to permit the expression of the heterologousDNA in the host cell. As described herein, such expression constuctsoptionally also contain nucleotide sequences which encode endosomaltargeting signals, preferably human invariant chain or a targetingfragment thereof.

Preferred systems for mRNA expression in mammalian cells are those suchas pRc/CMV and pcDNA3.1 (available from Invitrogen, Carlsbad, Calif.)that contain a selectable marker such as a gene that confers G418resistance (which facilitates the selection of stably transfected celllines) and the human cytomegalovirus (CMV) enhancer-promoter sequences.Additionally, suitable for expression in primate or canine cell lines isthe pCEP4 vector (Invitrogen), which contains an Epstein Barr virus(EBV) origin of replication, facilitating the maintenance of plasmid asa multicopy extrachromosomal element. Another expression vector is thepEF-BOS plasmid containing the promoter of polypeptide Elongation Factor1α which stimulates efficiently transcription in vitro. The plasmid isdescribed by Mishizuma and Nagata (Nuc. Acids Res. 18:5322, 1990), andits use in transfection experiments is disclosed by, for example,Demoulin (Mol. Cell. Biol. 16:4710-4716, 1996). Still another preferredexpression vector is an adenovirus, described by Stratford-Perricaudet,which is defective for E1 and E3 proteins (J. Clin. Invest. 90:626-630,1992). The use of the adenovirus as an Adeno.P1A recombinant isdisclosed by Warnier et al., in intradermal injection in mice forimmunization against P1A (Int. J. Cancer, 67:303-310, 1996). Recombinantvectors including viruses selected from the group consisting ofadenoviruses, adeno-associated viruses, poxviruses including vacciniaviruses and attenuated poxviruses such as ALVAC, NYVAC, Semliki Forestvirus, Venezuelan equine encephalitis virus, retroviruses, Sindbisvirus, Ty virus-like particle, other alphaviruses, VSV, plasmids (e.g.“naked” DNA), bacteria (e.g. the bacterium Bacille Calmette Guerin,attenuated Salmonella), and the like can be used in such delivery, forexample, for use as a vaccine.

The invention also embraces so-called expression kits, which allow theartisan to prepare a desired expression vector or vectors. Suchexpression kits include at least separate portions of at least two ofthe previously discussed materials. Other components may be added, asdesired.

The invention as described herein has a number of uses, some of whichare described herein. The following uses are described for MAGE HLAclass II binding peptides but are equally applicable to use of otherMAGE family HLA class II binding peptides. First, the invention permitsthe artisan to diagnose a disorder characterized by expression of a MAGEHLA class II binding peptide. These methods involve determiningexpression of a MAGE HLA class II binding peptide, or a complex of aMAGE HLA class II binding peptide and an HLA class II molecule in abiological sample. The expression of a peptide or complex of peptide andHLA class II molecule can be determined by assaying with a bindingpartner for the peptide or complex, such as an antibody.

The invention further includes nucleic acid or protein microarrays withcomponents that bind MAGE HLA class II peptides or nucleic acidsencoding such polypeptides. In this aspect of the invention, standardtechniques of microarray technology are utilized to assess expression ofthe MAGE polypeptides and/or identify biological constituents that bindsuch polypeptides. The constituents of biological samples includeantibodies, lymphocytes (particularly T lymphocytes), and the like.Protein microarray technology, which is also known by other namesincluding: protein chip technology and solid-phase protein arraytechnology, is well known to those of ordinary skill in the art and isbased on, but not limited to, obtaining an array of identified peptidesor proteins on a fixed substrate, binding target molecules or biologicalconstituents to the peptides, and evaluating such binding. See, e.g., G.MacBeath and S. L. Schreiber, “Printing Proteins as Microarrays forHigh-Throughput Function Determination,” Science 289(5485):1760-1763,2000. Polypeptide arrays, particularly arrays that bind MAGE peptides(e.g., including the TCR sequences disclosed herein) also can be usedfor diagnostic applications, such as for identifying subjects that havea condition characterized by MAGE polypeptide expression.

Microarray substrates include but are not limited to glass, silica,aluminosilicates, borosilicates, metal oxides such as alumina and nickeloxide, various clays, nitrocellulose, or nylon. The microarraysubstrates may be coated with a compound to enhance synthesis of a probe(peptide or nucleic acid) on the substrate. Coupling agents or groups onthe substrate can be used to covalently link the first nucleotide oramino acid to the substrate. A variety of coupling agents or groups areknown to those of skill in the art. Peptide or nucleic acid probes thuscan be synthesized directly on the substrate in a predetermined grid.Alternatively, peptide or nucleic acid probes can be spotted on thesubstrate, and in such cases the substrate may be coated with a compoundto enhance binding of the probe to the substrate. In these embodiments,presynthesized probes are applied to the substrate in a precise,predetermined volume and grid pattern, preferably utilizing acomputer-controlled robot to apply probe to the substrate in acontact-printing manner or in a non-contact manner such as ink jet orpiezo-electric delivery. Probes may be covalently linked to thesubstrate.

Targets are peptides or proteins and may be natural or synthetic. Thetissue may be obtained from a subject or may be grown in culture (e.g.from a cell line).

In some embodiments of the invention one or more control peptide orprotein molecules are attached to the substrate. Preferably, controlpeptide or protein molecules allow determination of factors such aspeptide or protein quality and binding characteristics, reagent qualityand effectiveness, binding success, and analysis thresholds and success.

In other embodiments, one or more control peptide or nucleic acidmolecules are attached to the substrate. Preferably, control nucleicacid molecules allow determination of factors such as bindingcharacteristics, reagent quality and effectiveness, hybridizationsuccess, and analysis thresholds and success.

Nucleic acid microarray technology, which is also known by other namesincluding: DNA chip technology, gene chip technology, and solid-phasenucleic acid array technology, is well known to those of ordinary skillin the art and is based on, but not limited to, obtaining an array ofidentified nucleic acid probes on a fixed substrate, labeling targetmolecules with reporter molecules (e.g., radioactive, chemiluminescent,or fluorescent tags such as fluorescein, Cye3-dUTP, or Cye5-dUTP),hybridizing target nucleic acids to the probes, and evaluatingtarget-probe hybridization. A probe with a nucleic acid sequence thatperfectly matches the target sequence will, in general, result indetection of a stronger reporter-molecule signal than will probes withless perfect matches. Many components and techniques utilized in nucleicacid microarray technology are presented in The Chipping Forecast,Nature Genetics, Vol.21, Jan 1999, the entire contents of which isincorporated by reference herein.

According to the present invention, nucleic acid microarray substratesmay include but are not limited to glass, silica, aluminosilicates,borosilicates, metal oxides such as alumina and nickel oxide, variousclays, nitrocellulose, or nylon. In all embodiments a glass substrate ispreferred. According to the invention, probes are selected from thegroup of nucleic acids including, but not limited to: DNA, genomic DNA,cDNA, and oligonucleotides; and may be natural or synthetic.Oligonucleotide probes preferably are 20 to 25-mer oligonucleotides andDNA/cDNA probes preferably are 500 to 5000 bases in length, althoughother lengths may be used. Appropriate probe length may be determined byone of ordinary skill in the art by following art-known procedures. Inone embodiment, preferred probes are sets of two or more molecule thatbind the nucleic acid molecules that encode the MAGE HLA class IIbinding peptides set forth herein. Probes may be purified to removecontaminants using standard methods known to those of ordinary skill inthe art such as gel filtration or precipitation.

In one embodiment, the microarray substrate may be coated with acompound to enhance synthesis of the probe on the substrate. Suchcompounds include, but are not limited to, oligoethylene glycols. Inanother embodiment, coupling agents or groups on the substrate can beused to covalently link the first nucleotide or oligonucleotide to thesubstrate. These agents or groups may include, for example, amino,hydroxy, bromo, and carboxy groups. These reactive groups are preferablyattached to the substrate through a hydrocarbyl radical such as analkylene or phenylene divalent radical, one valence position occupied bythe chain bonding and the remaining attached to the reactive groups.These hydrocarbyl groups may contain up to about ten carbon atoms,preferably up to about six carbon atoms. Alkylene radicals are usuallypreferred containing two to four carbon atoms in the principal chain.These and additional details of the process are disclosed, for example,in U.S. Pat. No. 4,458,066, which is incorporated by reference in itsentirety.

In one embodiment, probes are synthesized directly on the substrate in apredetermined grid pattern using methods such as light-directed chemicalsynthesis, photochemical deprotection, or delivery of nucleotideprecursors to the substrate and subsequent probe production.

In another embodiment, the substrate may be coated with a compound toenhance binding of the probe to the substrate. Such compounds include,but are not limited to: polylysine, amino silanes, amino-reactivesilanes (Chipping Forecast, 1999) or chromium. In this embodiment,presynthesized probes are applied to the substrate in a precise,predetermined volume and grid pattern, utilizing a computer-controlledrobot to apply probe to the substrate in a contact-printing manner or ina non-contact manner such as ink jet or piezo-electric delivery. Probesmay be covalently linked to the substrate with methods that include, butare not limited to, UV-irradiation. In another embodiment probes arelinked to the substrate with heat.

Targets for microarrays are nucleic acids selected from the group,including but not limited to: DNA, genomic DNA, cDNA, RNA, mRNA and maybe natural or synthetic. In all embodiments, nucleic acid targetmolecules from human tissue are preferred. The tissue may be obtainedfrom a subject or may be grown in culture (e.g. from a cell line).

In embodiments of the invention one or more control nucleic acidmolecules are attached to the substrate. Preferably, control nucleicacid molecules allow determination of factors such as nucleic acidquality and binding characteristics, reagent quality and effectiveness,hybridization success, and analysis thresholds and success. Controlnucleic acids may include but are not limited to expression products ofgenes such as housekeeping genes or fragments thereof.

In some embodiments, one or more control peptide or nucleic acidmolecules are attached to the substrate. Preferably, control nucleicacid molecules allow determination of factors such as bindingcharacteristics, reagent quality and effectiveness, hybridizationsuccess, and analysis thresholds and success.

The invention also permits the artisan to treat a subject having adisorder characterized by expression of a MAGE HLA class II bindingpeptide. Treatments include administering an agent which enriches in thesubject a complex of a MAGE HLA class II binding peptide and an HLAclass II molecule, and administering CD4⁺ T lymphocytes which arespecific for such complexes including T cells transfected or transducedto express T cell receptors that include the TCR sequences disclosedherein. Agents useful in the foregoing treatments include MAGE HLA classII binding peptides and functional variants thereof, endosome-targetedfusion proteins which include such MAGE peptides, nucleic acids whichexpress such proteins and peptides (including viruses which contain thenucleic acids), complexes of such peptides and HLA class II bindingmolecules (e.g. HLA DR1), antigen presenting cells bearing complexes ofa MAGE HLA class II binding peptide and an HLA class II bindingmolecule, and the like. The invention also permits an artisan toselectively enrich a population of T lymphocytes for CD4⁺ T lymphocytesspecific for a MAGE HLA class II binding peptide, for example byexposing a population of cells to a complex of a MAGE HLA class IIbinding peptide and an HLA class II binding molecule.

The isolation of the MAGE HLA class II binding peptides also makes itpossible to isolate nucleic acids which encode the MAGE HLA class IIbinding peptides. Nucleic acids can be used to produce in vitro or inprokaryotic or eukaryotic host cells the MAGE HLA class II bindingpeptides. A variety of methodologies well-known to the skilledpractitioner can be utilized to obtain isolated MAGE HLA class IIbinding peptides. For example, an expression vector may be introducedinto cells to cause production of the peptides. In another method, mRNAtranscripts may be microinjected or otherwise introduced into cells tocause production of the encoded peptides. Translation of mRNA incell-free extracts such as the reticulocyte lysate system also may beused to produce peptides. Peptides comprising the MAGE HLA class IIbinding peptide of the invention may also be synthesized in vitro. Thoseskilled in the art also can readily follow known methods for isolatingpeptides in order to obtain isolated MAGE HLA class II binding peptides.These include, but are not limited to, immunochromatography, HPLC,size-exclusion chromatography, ion-exchange chromatography andimmune-affinity chromatography.

These isolated MAGE HLA class II binding peptides, proteins whichinclude such peptides, or complexes (including soluble complexes such astetramers) of the peptides and HLA class II molecules, such as HLA-DR1molecules, may be combined with materials such as adjuvants to producevaccines useful in treating disorders characterized by expression of theMAGE HLA class II binding peptide. In addition, vaccines can be preparedfrom cells which present the MAGE HLA class II binding peptide/HLAcomplexes on their surface, such as dendritic cells, B cells,non-proliferative transfectants, etcetera. In all cases where cells areused as a vaccine, these can be cells transfected with coding sequencesfor one or both of the components necessary to stimulate CD4⁺lymphocytes, or can be cells which already express both moleculeswithout the need for transfection. For example, autologous antigenpresenting cells can be isolated from a patient and treated to obtaincells which present MAGE epitopes in association of HLA class I and HLAclass II molecules. These cells would be capable of stimulating bothCD4⁺ and CD8⁺ cell responses. Such antigen presenting cells can beobtained by infecting dendritic cells with recombinant viruses encodingan Ii.MAGE fusion protein. Dendritic cells also can be loaded with HLAclass I and HLA class II epitopes.

Vaccines also encompass naked DNA or RNA, encoding a MAGE HLA class IIbinding peptide or precursor thereof, which may be produced in vitro andadministered via injection, particle bombardment, nasal aspiration andother methods. Vaccines of the “naked nucleic acid” type have beendemonstrated to provoke an immunological response including generationof CTLs specific for the peptide encoded by the naked nucleic acid(Science 259:1745-1748, 1993). Vaccines also include nucleic acidspackaged in a virus, liposome or other particle, including polymericparticles useful in drug delivery.

The immune response generated or enhanced by any of the treatmentsdescribed herein can be monitored by various methods known in the art.For example, the presence of T cells specific for a given antigen can bedetected by direct labeling of T cell receptors with soluble fluorogenicMHC molecule tetramers which present the antigenic peptide (Altman etal., Science 274:94-96, 1996; Dunbar et al., Curr. Biol. 8:413-416,1998). Briefly, soluble MHC class I molecules are folded in vitro in thepresence of β2-microglobulin and a peptide antigen which binds the classI molecule. After purification, the MHC/peptide complex is purified andlabeled with biotin. Tetramers are formed by mixing the biotinylatedpeptide-MHC complex with labeled avidin (e.g. phycoerythrin) at a molarratio of 4:1. Tetramers are then contacted with a source of CTLs such asperipheral blood or lymph node. The tetramers bind CTLs which recognizethe peptide antigen/MHC class I complex. Cells bound by the tetramerscan be sorted by fluorescence activated cell sorting to isolate thereactive CTLs. The isolated CTLs then can be expanded in vitro for useas described herein. The use of MHC class II molecules as tetramers wasrecently demonstrated by Crawford et al. (Immunity 8:675-682, 1998; seealso Dunbar and Ogg, J. Immunol. Methods 268(1):3-7, 2002; Arnold etal., J. Immunol. Methods 271(1-2):137-151, 2002). Multimeric soluble MHCclass II molecules were complexed with a covalently attached peptide(which can be attached with or without a linker molecule), but also canbe loaded onto class II molecules. The class II tetramers were shown tobind with appropriate specificity and affinity to specific T cells. Thustetramers can be used to monitor both CD4⁺ and CD8+ cell responses tovaccination protocols. Methods for preparation of multimeric complexesof MHC class II molecules are described in Hugues et al., J.Immunological Meth. 268: 83-92, (2002) and references cited therein,each of which is incorporated by reference.

The MAGE HLA class II binding peptide, as well as complexes of MAGE HLAclass II binding peptide and HLA molecule, also may be used to produceantibodies, using standard techniques well known to the art. Standardreference works setting forth the general principles of antibodyproduction include Catty, D., Antibodies, A Practical Approach, Vol. 1,IRL Press, Washington D.C. (1988); Klein, J., Immunology: The Science ofCell-Non-Cell Discrimination, John Wiley and Sons, New York (1982);Kennett, R., et al., Monoclonal Antibodies, Hybridoma, A New DimensionIn Biological Analyses, Plenum Press, New York (1980); Campbell, A.,Monoclonal Antibody Technology, in Laboratory Techniques andBiochemistry and Molecular Biology, Vol. 13 (Burdon, R. et al. EDS.),Elsevier Amsterdam (1984); and Eisen, H. N., Microbiology, thirdedition, Davis, B. D. et al. EDS. (Harper & Rowe, Philadelphia (1980).

Significantly, as is well-known in the art, only a small portion of anantibody molecule, the paratope, is involved in the binding of theantibody to its epitope (see, in general, Clark, W. R. (1986) TheExperimental Foundations of Modern Immunology Wiley & Sons, Inc., NewYork; Roitt, I. (1991) Essential Immunology, 7th Ed., BlackwellScientific Publications, Oxford). The pFc′ and Fe regions, for example,are effectors of the complement cascade but are not involved in antigenbinding. An antibody from which the pFc′ region has been enzymaticallycleaved, or which has been produced without the pFc′ region, designatedan F(ab′)₂ fragment, retains both of the antigen binding sites of anintact antibody. Similarly, an antibody from which the Fc region hasbeen enzymatically cleaved, or which has been produced without the Fcregion, designated an Fab fragment, retains one of the antigen bindingsites of an intact antibody molecule. Proceeding further, Fab fragmentsconsist of a covalently bound antibody light chain and a portion of theantibody heavy chain denoted Fd. The Fd fragments are the majordeterminant of antibody specificity (a single Fd fragment may beassociated with up to ten different light chains without alteringantibody specificity) and Fd fragments retain epitope-binding ability inisolation.

Within the antigen-binding portion of an antibody, as is well-known inthe art, there are complementarity determining regions (CDRs), whichdirectly interact with the epitope of the antigen, and framework regions(FRs), which maintain the tertiary structure of the paratope (see, ingeneral, Clark, 1986; Roitt, 1991). In both the heavy chain Fd fragmentand the light chain of IgG immunoglobulins, there are four frameworkregions (FR1 through FR4) separated respectively by threecomplementarity determining regions (CDR1 through CDR3). The CDRs, andin particular the CDR3 regions, and more particularly the heavy chainCDR3, are largely responsible for antibody specificity.

It is now well-established in the art that the non-CDR regions of amammalian antibody may be replaced with similar regions of conspecificor heterospecific antibodies while retaining the epitopic specificity ofthe original antibody. This is most clearly manifested in thedevelopment and use of “humanized” antibodies in which non-human CDRsare covalently joined to human FR and/or Fc/pFc′ regions to produce afunctional antibody. See, e.g., U.S. Pat. Nos. 4,816,567, 5,225,539,5,585,089, 5,693,762 and 5,859,205.

Fully human monoclonal antibodies also can be prepared by immunizingmice transgenic for large portions of human immunoglobulin heavy andlight chain loci. See, e.g., U.S. Pat. Nos. 5,545,806, 6,150,584, andreferences cited therein. Following immunization of these mice (e.g.,XenoMouse (Abgenix), HuMAb mice (Medarex/GenPharm)), monoclonalantibodies can be prepared according to standard hybridoma technology.These monoclonal antibodies will have human immunoglobulin amino acidsequences and therefore will not provoke human anti-mouse antibody(HAMA) responses when administered to humans.

Thus, as will be apparent to one of ordinary skill in the art, thepresent invention also provides for F(ab′)₂, Fab, Fv and Fd fragments;chimeric antibodies in which the Fc and/or FR and/or CDR1 and/or CDR2and/or light chain CDR3 regions have been replaced by homologous humanor non-human sequences; chimeric F(ab′)₂ fragment antibodies in whichthe FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have beenreplaced by homologous human or non-human sequences; chimeric Fabfragment antibodies in which the FR and/or CDR1 and/or CDR2 and/or lightchain CDR3 regions have been replaced by homologous human or non-humansequences; and chimeric Fd fragment antibodies in which the FR and/orCDR1 and/or CDR2 regions have been replaced by homologous human ornon-human sequences. The present invention also includes so-calledsingle chain antibodies.

Methods for identifying Fab molecules endowed with the antigen-specific,HLA-restricted specificity of T cells has been described by Denkberg etal. Proc. Nat'l. Acad. Sci. USA 99:9421-9426 (2002) and Cohen et al.Cancer Res. 62:5835-5844 (2002), both of which are incorporated hereinby reference. Methods for generating and identifying other antibodymolecules, e.g., scFv and diabodies are well known in the art, see e.g.,Bird et al., Science, 242:423-426 (1988); Huston et al., Proc. Nat'l.Acad. Sci. USA 85:5879-5883 (1988); Mallender and Voss, J. Biol. Chem.269:199-206 (1994); Ito and Kurosawa, J. Biol. Chem. 27: 20668-20675(1993), and Gandecha et al., Prot. Express. Purif 5: 385-390 (1994).

The antibodies of the present invention thus are prepared by any of avariety of methods, including administering protein, fragments ofprotein, cells expressing the protein or fragments thereof and anappropriate HLA class II molecule, and the like to an animal to inducepolyclonal antibodies. The production of monoclonal antibodies isaccording to techniques well known in the art. Binding molecules canalso be identified by screening libraries of binding peptides (e.g.,phage display libraries); the binding molecules can be incorporatedrecombinantly into antibody or TCR molecules using standardmethodologies.

The antibodies of this invention can be used for experimental purposes(e.g., localization of the HLA/peptide complexes, immunoprecipitations,Western blots, flow cytometry, ELISA etc.) as well as diagnostic ortherapeutic purposes (e.g., assaying extracts of tissue biopsies for thepresence of HLA/peptide complexes, targeting delivery of cytotoxic orcytostatic substances to cells expressing the appropriate HLA/peptidecomplex). The antibodies of this invention are useful for the study andanalysis of antigen presentation on tumor cells and can be used to assayfor changes in the HLA/peptide complex expression before, during orafter a treatment protocol, e.g., vaccination with peptides, antigenpresenting cells, HLA/peptide tetramers, adoptive transfer orchemotherapy.

The antibodies and antibody fragments of this invention may be coupledto diagnostic labeling agents for imaging of cells and tissues thatexpress the HLA/peptide complexes or may be coupled to therapeuticallyuseful agents by using standard methods well-known in the art. Theantibodies also may be coupled to labeling agents for imaging e.g.,radiolabels or fluorescent labels, or may be coupled to, e.g., biotin orantitumor agents, e.g., radioiodinated compounds, toxins such as ricin,methotrexate, cytostatic or cytolytic drugs, etc. Examples of diagnosticagents suitable for conjugating to the antibodies of this inventioninclude e.g., barium sulfate, diatrizoate sodium, diatrizoate meglumine,iocetamic acid, iopanoic acid, ipodate calcium, metrizamide, tyropanoatesodium and radiodiagnostics including positron emitters such asfluorine-18 and carbon-11, gamma emitters such as iodine-123,technitium-99m, iodine-131 and indium-111, nuclides for nuclear magneticresonance such as fluorine and gadolinium. As used herein,“therapeutically useful agents” include any therapeutic molecules, whichare preferably targeted selectively to a cell expressing the HLA/peptidecomplexes, including antineoplastic agents, radioiodinated compounds,toxins, other cytostatic or cytolytic drugs. Antineoplastic therapeuticsare well known and include: aminoglutethimide, azathioprine, bleomycinsulfate, busulfan, carmustine, chlorambucil, cisplatin,cyclophosphamide, cyclosporine, cytarabidine, dacarbazine, dactinomycin,daunorubicin, doxorubicin, taxol, etoposide, fluorouracil,interferon-.alpha., lomustine, mercaptopurine, methotrexate, mitotane,procarbazine HCl, thioguanine, vinblastine sulfate and vincristinesulfate. Additional antineoplastic agents include those disclosed inChapter 52, Antineoplastic Agents (Paul Calabresi and Bruce A. Chabner),and the introduction thereto, 1202-1263, of Goodman and Gilman's “ThePharmacological Basis of Therapeutics”, Eighth Edition, 1990,McGraw-Hill, Inc. (Health Professions Division). Toxins can be proteinssuch as, for example, pokeweed anti-viral protein, cholera toxin,pertussis toxin, ricin, gelonin, abrin, diphtheria exotoxin, orPseudomonas exotoxin. Toxin moieties can also be high energy-emittingradionuclides such as cobalt-60. The antibodies may be administered to asubject having a pathological condition characterized by thepresentation of the HLA/peptide complexes of this invention, e.g.,melanoma or other cancers, in an amount sufficient to alleviate thesymptoms associated with the pathological condition.

Soluble T cell receptors (TCRs) which specifically bind to theHLA/peptide complexes described herein are also an aspect of thisinvention. In their soluble form, T cell receptors are analogous to amonoclonal antibody in that they bind to HLA/peptide complex in apeptide-specific manner. Immobilized TCRs or antibodies may be used toidentify and purify unknown peptide/HLA complexes which may be involvedin cellular abnormalities. Methods for identifying and isolating solubleTCRs are known in the art, see for example WO 99/60119, WO 99/60120(both incorporated herein by reference) which describe syntheticmultivalent T cell receptor complexes for binding to peptide-MHCcomplexes. Recombinant, refolded soluble T cell receptors arespecifically described. Such receptors may be used for deliveringtherapeutic agents or detecting specific peptide-MHC complexes expressedby tumor cells. WO 02/088740 (incorporated by reference) describes amethod for identifying a substance that binds to a peptide-MHC complex.A peptide-MHC complex is formed between a predetermined MHC and peptideknown to bind to such predetermined MHC. The complex is then use toscreen or select an entity that binds to the peptide-MHC complex such asa T cell receptor. The method could also be applied to the selection ofmonoclonal antibodies that bind to the predetermined peptide-MHCcomplex.

The invention also includes molecules that include or are made using theT cell receptor (TCR) sequences disclosed herein. Such molecules includesynthetic multivalent complexes of TCRs, soluble TCRs, and the like. Theinvention thus provides synthetic multivalent T cell receptor complexesthat can be used for binding to a MHC-peptide complex. The TCR complexesinclude a plurality of T cell receptors specific for the MHC-peptidecomplexes disclosed herein (i.e., those that include the MAGE HLA classII binding peptides disclosed herein). In preferred embodiments, the Tcell receptors are those disclosed herein, or alternatively include asequences from the TCRs disclosed herein, most preferably the CDR3regions. Preferred T cell receptor sequences comprising a CDR3 sequenceinclude SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ IDNO:59, and SEQ ID NO:62. TCRs present in complexes and soluble formpreferably include an α chain and a β chain. As shown below, three pairsof α and β chains were cloned and sequenced; these contain CDR3sequences selected from the group consisting of (1) SEQ ID NO:47 and SEQID NO:50; (2) SEQ ID NO:53 and SEQ ID NO:56; and SEQ ID NO:59 and SEQ IDNO:62. Recombinant TCRs, including those that include a CDR3 regiondisclosed herein, can be produced recombinantly using standardtechniques of molecular biology well known to those of ordinary skill inthe art. Preparation of TCRs and TCR complexes is known in the art; see,e.g., published international patent applications WO 99/60119 and WO99/60120, each of which is incorporated herein by reference.

The TCR molecules, including α and β chains, can be prepared as singlechain molecules or can be associated using a dimerization molecule suchas a leucine zipper, a coiled coil domain, immunoglobulin domains orother molecules that permit dimerization, preferably heterodimerization.

In preferred forms, the synthetic multivalent TCR complexes provided inaccordance with the invention are tetrameric complexes. In the TCRcomplexes provided herein, T cell receptors are associated with oneanother via a linker molecule, which can be a multivalent attachmentmolecule (a “hub” molecule) that provides multiple binding sites for theTCRs, such as avidin or streptavidin that will bind four biotinylatedTCR molecules.

Synthetic multivalent TCR complexes and soluble TCRs are useful forbinding to MHC-peptide complexes, and thus can be used for therapeuticand diagnostic or prognostic purposes, including those described hereinin connection with antibodies. Thus in some instances the TCR complexcan include a detectable label, such as a fluorophore, a chromophore, ora radioactive molecule. In other embodiments, the TCR complex caninclude a therapeutic agent, such as a cytotoxic agent or animmunostimulating agent. For therapeutic and/or diagnostic usesinvolving adminstration of TCR complexes, the complex will preferably beprepared in a pharmaceutically acceptable formulation in accordance withstandard practice in the pharmaceutical arts.

Also an embodiment of this invention are nucleic acid molecules encodingthe antibodies and T cell receptors of this invention and host cells,e.g., human T cells, transformed with a nucleic acid molecule encoding arecombinant antibody or antibody fragment, e.g., scFv or Fab, or a TCRspecific for a predesignated HLA/peptide complex as described herein.Recombinant Fab or TCR specific for a predesignated HLA/peptide complexin T cells have been described in, e.g., Chung et al., Proc. Nat'l.Acad. Sci. USA 91(26):12654-12658 (1994); Willemsen et al., Gene Ther.8(21):1601-1608 (2001); Willemsen et al., Gene Ther. 7(16):1369-1377(2000), Willemsen et al., Hum. Immunol. 64(1):56-68 (2003); Schaft etal., J. Immunol. 170(4):2186-2194 (2003); and Fujio et al., J. Immunol.165(1):528-532 (2000) (each of which is incorporated herein byreference) and have applications in an autologous T cell transfersetting. The autologous T cells, transduced to express recombinantantibody or TCR, may be infused into a patient having a pathologicalcondition associated with cells expressing the HLA/peptide complex,particularly cancer. The transduced T cells are administered in anamount sufficient to inhibit the progression or alleviate at least someof the symptoms associated with the pathological condition.

Thus the invention also provides T lymphocytes that are modified toinclude at least one T cell receptor sequence that comprises a CDR3sequence (e.g., by transfection or transduction of nucleic acidmolecules that encode TCR sequences) and methods of making such Tlymphocytes. The T lymphocytes can be used therapeutically to provide animmune response for a subject that has a cancer wherein MAGE proteinsare expressed, for example, to ameliorate the cancer, includingreduction of disease symptoms. To prepare the modified T lymphocytes,peripheral blood cells are isolated from a subject, then transfected ortransduced with one or more vectors that expresses at least one T cellreceptor sequence that specifically binds a MAGE HLA class II bindingpeptide bound to a HLA class II molecule, preferably HLA-DRB1. The Tcell receptor sequence preferably includes a CDR3 sequence selected fromthe group consisting of SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ IDNO:56, SEQ ID NO:59, and SEQ ID NO:62. The vectors used for T lymphocytemodification typically are expression plasmids or viruses, particularlyretroviruses. It is preferred that the one or more vectors express an αchain and a β chain, or a single chain TCR.

The transfected or transduced cells then are administered to the subjectin an amount effective to increase an immune response or to amelioratethe cancer. The T cells can be expanded in vitro prior toadministration. In certain embodiments, the TCR sequence includes atleast one CDR3 sequence selected from the group consisting of SEQ IDNO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, and SEQID NO:62.

When “disorder” or “condition” is used herein, it refers to anypathological condition where the MAGE HLA class II binding peptide isexpressed. Such disorders include cancers, such as biliary tract cancer;bladder cancer; breast cancer; brain cancer including glioblastomas andmedulloblastomas; cervical cancer; choriocarcinoma; colon cancerincluding colorectal carcinomas; endometrial cancer; esophageal cancer;gastric cancer; head and neck cancer; hematological neoplasms includingacute lymphocytic and myelogenous leukemia, multiple myeloma,AIDS-associated leukemias and adult T-cell leukemia lymphoma;intraepithelial neoplasms including Bowen's disease and Paget's disease;liver cancer; lung cancer including small cell lung cancer and non-smallcell lung cancer; lymphomas including Hodgkin's disease and lymphocyticlymphomas; neuroblastomas; oral cancer including squamous cellcarcinoma; osteosarcomas; ovarian cancer including those arising fromepithelial cells, stromal cells, germ cells and mesenchymal cells;pancreatic cancer; prostate cancer; rectal cancer; sarcomas includingleiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma, synovialsarcoma and osteosarcoma; skin cancer including melanomas, Kaposi'ssarcoma, basocellular cancer, and squamous cell cancer; testicularcancer including germinal tumors such as seminoma, non-seminoma(teratomas, choriocarcinomas), stromal tumors, and germ cell tumors;thyroid cancer including thyroid adenocarcinoma and medullar carcinoma;transitional cancer and renal cancer including adenocarcinoma and Wilmstumor.

Some therapeutic approaches based upon the disclosure are premised oninducing a response by a subject's immune system to MAGE HLA class IIbinding peptide presenting cells. One such approach is theadministration of autologous CD4⁺ T cells specific to the complex ofMAGE HLA class II binding peptide and an HLA class II molecule to asubject with abnormal cells of the phenotype at issue. It is within theskill of the artisan to develop such CD4⁺ T cells in vitro. Generally, asample of cells taken from a subject, such as blood cells, are contactedwith a cell presenting the complex and capable of provoking CD4⁺ Tlymphocytes to proliferate; alternatively, T cells of appropriatespecificity can be sorted from a larger population of T cells using anHLA-MAGE peptide multimer (e.g., tetramer). The target cell can be atransfectant, such as a COS cell, or an antigen presenting cell bearingHLA class II molecules, such as dendritic cells or B cells. Thesetransfectants present the desired complex of their surface and, whencombined with a CD4⁺ T lymphocyte of interest, stimulate itsproliferation. COS cells are widely available, as are other suitablehost cells. Specific production of CD4⁺ T lymphocytes is describedbelow. The clonally expanded autologous CD4⁺ T lymphocytes then areadministered to the subject. The CD4⁺ T lymphocytes then stimulate thesubject's immune response, thereby achieving the desired therapeuticgoal.

CD4⁺ T cells specific to a complex of a MAGE HLA class II bindingpeptide and an HLA class II molecule also can be prepared bytransfecting or transducing T lymphocytes with T cell receptor sequencesincluding the CDR sequences described herein, as noted above.

CTL proliferation can be increased by increasing the level of tryptophanin T cell cultures, by inhibiting enzymes which catabolizes tryptophan,such as indoleamine 2,3-dioxygenase (IDO), or by adding tryptophan tothe culture (see, e.g., PCT application WO99/29310). Proliferation of Tcells is enhanced by increasing the rate of proliferation and/orextending the number of divisions of the T cells in culture. Inaddition, increasing tryptophan in T cell cultures also enhances thelytic activity of the T cells grown in culture.

The foregoing therapy assumes that at least some of the subject'sabnormal cells present the relevant HLA/peptide complex. This can bedetermined very easily, as the art is very familiar with methods foridentifying cells which present a particular HLA molecule, as well ashow to identify cells expressing DNA of the pertinent sequences, in thiscase a MAGE sequence.

The foregoing therapy is not the only form of therapy that is availablein accordance with the invention. CD4⁺ T lymphocytes can also beprovoked in vivo, using a number of approaches. One approach is the useof non-proliferative cells expressing the complex. The cells used inthis approach may be those that normally express the complex, such asdendritic cells or cells transfected with one or both of the genesnecessary for presentation of the complex. Chen et al., (Proc. Natl.Acad. Sci. USA 88: 110-114, 1991) exemplifies this approach, showing theuse of transfected cells expressing HPV-E7 peptides in a therapeuticregime. Various cell types may be used. Similarly, vectors carrying oneor both of the genes of interest may be used. Viral or bacterial vectorsare especially preferred. For example, nucleic acids which encode a MAGEHLA class II binding peptide may be operably linked to promoter andenhancer sequences which direct expression of the MAGE HLA class IIbinding peptide in certain tissues or cell types. The nucleic acid maybe incorporated into an expression vector. Expression vectors may beunmodified extrachromosomal nucleic acids, plasmids or viral genomesconstructed or modified to enable insertion of exogenous nucleic acids,such as those encoding MAGE HLA class II binding peptides. Nucleic acidsencoding a MAGE HLA class II binding peptide also may be inserted into aretroviral genome, thereby facilitating integration of the nucleic acidinto the genome of the target tissue or cell type. In these systems, thegene of interest is carried by a microorganism, e.g., a vaccinia virus,poxviruses in general, adenovirus, herpes simplex virus, retrovirus orthe bacteria BCG, and the materials de facto “infect” host cells. Thecells which result present the complex of interest, and are recognizedby autologous CD4⁺ T cells, which then proliferate.

A similar effect can be achieved by combining a MAGE HLA class IIbinding peptide with an adjuvant to facilitate incorporation into HLAclass II presenting cells in vivo. If larger than the HLA class IIbinding portion, the MAGE HLA class II binding peptide can be processedif necessary to yield the peptide partner of the HLA molecule while theTRA is presented without the need for further processing. Generally,subjects can receive an intradermal injection of an effective amount ofthe MAGE HLA class II binding peptide. Initial doses can be followed bybooster doses, following immunization protocols standard in the art.

A preferred method for facilitating incorporation of MAGE HLA class IIbinding peptides into HLA class II presenting cells is by expressing inthe presenting cells a polypeptide which includes an endosomal targetingsignal fused to a MAGE polypeptide which includes the class II bindingpeptide. Particularly preferred are MAGE fusion proteins which containhuman invariant chain Ti.

Any of the foregoing compositions or protocols can include also MAGE HLAclass I binding peptides for induction of a cytolytic T lymphocyteresponse. For example, the MAGE-A3 protein can be processed in a cell toproduce both HLA class I and HLA class II responses. Several suchpeptides have been described in U.S. Pat. Nos. 5,585,461 and 5,591,430,and PCT published application PCT/US95/03657, as well as by Gaugler etal. (J. Exp. Mecl. 179:921-930, 1994), van der Bruggen et al. (Eur. J.Immunol. 24:3038-3043, 1994), and Herman et al. (Immunogenetics43:377-383, 1996). By administering MAGE peptides which bind HLA class Iand class II molecules (or nucleic acid encoding such peptides), animproved immune response may be provided by inducing both T helper cellsand T killer cells (CTLs).

In addition, non-MAGE tumor associated peptides also can be administeredto increase immune response via HLA class I and/or class II. It is wellestablished that cancer cells can express more that one tumor associatedgene. It is within the scope of routine experimentation for one ofordinary skill in the art to determine whether a particular subjectexpresses additional tumor associated genes, and then include HLA classI and/or HLA class II binding peptides derived from expression productsof such genes in the foregoing MAGE compositions and vaccines.

Especially preferred are nucleic acids encoding a series of epitopes,known as “polytopes”. The epitopes can be arranged in sequential oroverlapping fashion (see, e.g., Thomson et al., Proc. Natl. Acad. Sci.USA 92:5845-5849, 1995; Gilbert et al., Nature Biotechnol. 15:1280-1284,1997), with or without the natural flanking sequences, and can beseparated by unrelated linker sequences if desired. The polytope isprocessed to generated individual epitopes which are recognized by theimmune system for generation of immune responses.

Thus, for example, MAGE HLA class II binding peptides can be combinedwith peptides from other tumor rejection antigens (e.g. by preparationof hybrid nucleic acids or polypeptides) and with MAGE HLA class Ibinding peptides (some of which are listed below) to form “polytopes”.Exemplary tumor associated peptide antigens that can be administered toinduce or enhance an immune response are derived from tumor associatedgenes and encoded proteins including MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4,MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11,MAGE-A12, MAGE-A13, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6,GAGE-7, GAGE-8, BAGE-1, RAGE-1, LB33/MUM-1, PRAME, NAG, MAGE-Xp2(MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), tyrosinase, brainglycogen phosphorylase, Melan-A, MAGE-C1 (CT-7), MAGE-C2 (CT-10),NY-ESO-1, LAGE-1, SSX-1, SSX-2 (HOM-MEL-40), SSX-4, SSX-5, and SCP-1.For example, antigenic peptides characteristic of tumors include thoselisted in published PCT application WO 00/20581 (PCT/US99/21230).

Other examples of HLA class I and HLA class II binding peptides will beknown to one of ordinary skill in the art (for example, see Coulie, StemCells 13:393-403, 1995; there is a listing on the website of the journalCancer Immunity in the “Peptide Database” section,http://www.cancerimmunity.org/peptidedatabase/Tcellepitopes.htm), andcan be used in the invention in a like manner as those disclosed herein.One of ordinary skill in the art can prepare polypeptides comprising oneor more MAGE peptides and one or more of the foregoing tumor rejectionpeptides, or nucleic acids encoding such polypeptides, according tostandard procedures of molecular biology.

Thus polytopes are groups of two or more potentially immunogenic orimmune response stimulating peptides which can be joined together invarious arrangements (e.g. concatenated, overlapping). The polytope (ornucleic acid encoding the polytope) can be administered in a standardimmunization protocol, e.g. to animals, to test the effectiveness of thepolytope in stimulating, enhancing and/or provoking an immune response.

The peptides can be joined together directly or via the use of flankingsequences to form polytopes, and the use of polytopes as vaccines iswell known in the art (see, e.g., Thomson et al., Proc. Acad. Natl.Acad. Sci USA 92(13):5845-5849, 1995; Gilbert et al., Nature Biotechnol.15(12):1280-1284, 1997; Thomson et al., J. Immunol. 157(2):822-826,1996; Tam et al., J. Exp. Med. 171(1):299-306, 1990). For example, Tamshowed that polytopes consisting of both MHC class I and class IIbinding epitopes successfully generated antibody and protective immunityin a mouse model. Tam also demonstrated that polytopes comprising“strings” of epitopes are processed to yield individual epitopes whichare presented by MHC molecules and recognized by CTLs. Thus polytopescontaining various numbers and combinations of epitopes can be preparedand tested for recognition by CTLs and for efficacy in increasing animmune response.

It is known that tumors express a set of tumor antigens, of which onlycertain subsets may be expressed in the tumor of any given patient.Polytopes can be prepared which correspond to the different combinationof epitopes representing the subset of tumor rejection antigensexpressed in a particular patient. Polytopes also can be prepared toreflect a broader spectrum of tumor rejection antigens known to beexpressed by a tumor type. Polytopes can be introduced to a patient inneed of such treatment as polypeptide structures, or via the use ofnucleic acid delivery systems known in the art (see, e.g., Allsopp etal., Eur. J. Immunol. 26(8):1951-1959, 1996). Adenovirus, pox virus,Ty-virus like particles, adeno-associated virus, plasmids, bacteria,etc. can be used in such delivery. One can test the polytope deliverysystems in mouse models to determine efficacy of the delivery system.The systems also can be tested in human clinical trials.

As part of the immunization compositions, one or more substances thatpotentiate an immune response are administered along with the peptidesdescribed herein. Such substances include adjuvants and cytokines. Anadjuvant is a substance incorporated into or administered with antigenwhich potentiates the immune response. Adjuvants may enhance theimmunological response by providing a reservoir of antigen(extracellularly or within macrophages), activating macrophages andstimulating specific sets of lymphocytes. Adjuvants of many kinds arewell known in the art. Specific examples of adjuvants includemonophosphoryl lipid A (MPL, SmithKline Beecham), a congener obtainedafter purification and acid hydrolysis of Salmonella minnesota Re 595lipopolysaccharide; saponins including QS21 (SmithKline Beecham), a pureQA-21 saponin purified from Quillja saponaria extract; DQS21, describedin PCT application WO96/33739 (SmithKline Beecham); QS-7, QS-17, QS-18,and QS-L1 (So et al., Mol. Cells 7:178-186, 1997); incomplete Freund'sadjuvant; complete Freund's adjuvant; montanide; immunostimulatoryoligonucleotides (see e.g. CpG oligonucleotides described by Kreig etal., Nature 374:546-9, 1995); reagents that bind to one of the toll-likereceptors; vitamin E and various water-in-oil emulsions prepared frombiodegradable oils such as squalene and/or tocopherol. Preferably, thepeptides are administered mixed with a combination of DQS21/MPL. Theratio of DQS21 to MPL typically will be about 1:10 to 10:1, preferablyabout 1:5 to 5:1 and more preferably about 1:1. Typically for humanadministration, DQS21 and MPL will be present in a vaccine formulationin the range of about 1 μg to about 100 μg. Other adjuvants are known inthe art and can be used in the invention (see, e.g. Goding, MonoclonalAntibodies: Principles and Practice, 2nd Ed., 1986). Methods for thepreparation of mixtures or emulsions of peptide and adjuvant are wellknown to those of skill in the art of vaccination.

Other agents which stimulate the immune response of the subject can alsobe administered to the subject. For example, other cytokines are alsouseful in vaccination protocols as a result of their lymphocyteregulatory properties. Many other cytokines useful for such purposeswill be known to one of ordinary skill in the art, includinginterleukin-12 (IL-12) which has been shown to enhance the protectiveeffects of vaccines (see, e.g., Science 268: 1432-1434, 1995), GM-CSFand IL-18. Thus cytokines can be administered in conjunction withantigens and adjuvants to increase the immune response to the antigens.There are a number of additional immune response potentiating compoundsthat can be used in vaccination protocols. These include costimulatorymolecules provided in either protein or nucleic acid form. Suchcostimulatory molecules include the B7-1 and B7-2 (CD80 and CD86respectively) molecules which are expressed on dendritic cells (DC) andinteract with the CD28 molecule expressed on the T cell. Thisinteraction provides costimulation (signal 2) to an antigen/MHC/TCRstimulated (signal 1) T cell, increasing T cell proliferation andeffector function. B7 also interacts with CTLA4 (CD152) on T cells andstudies involving CTLA4 and B7 ligands indicate that the B7-CTLA4interaction can enhance antitumor immunity and CTL proliferation (Zhenget al., Proc. Nat'l Acad. Sci. USA 95:6284-6289, 1998).

B7 typically is not expressed on tumor cells so they are not efficientantigen presenting cells (APCs) for T cells. Induction of B7 expressionwould enable the tumor cells to stimulate more efficiently CTLproliferation and effector function. A combination of B7/IL-6/IL-12costimulation has been shown to induce IFN-gamma and a Th1 cytokineprofile in the T cell population leading to further enhanced T cellactivity (Gajewski et al., J. Immunol. 154:5637-5648, 1995). Tumor celltransfection with B7 has been discussed in relation to in vitro CTLexpansion for adoptive transfer immunotherapy by Wang et al. (J.Immunother. 19:1-8, 1996). Other delivery mechanisms for the B7 moleculewould include nucleic acid (naked DNA) immunization (Kim et al., NatureBiotechnol. 15:7:641-646, 1997) and recombinant viruses such as adenoand pox (Wendtner et al., Gene Ther. 4:726-735, 1997). These systems areall amenable to the construction and use of expression cassettes for thecoexpression of B7 with other molecules of choice such as the antigensor fragment(s) of antigens discussed herein (including polytopes) orcytokines. These delivery systems can be used for induction of theappropriate molecules in vitro and for in vivo vaccination situations.The use of anti-CD28 antibodies to directly stimulate T cells in vitroand in vivo could also be considered. Similarly, the inducibleco-stimulatory molecule ICOS which induces T cell responses to foreignantigen could be modulated, for example, by use of anti-ICOS antibodies(Hutloff et al., Nature 397:263-266, 1999).

Lymphocyte function associated antigen-3 (LFA-3) is expressed on APCsand some tumor cells and interacts with CD2 expressed on T cells. Thisinteraction induces T cell IL-2 and IFN-gamma production and can thuscomplement but not substitute, the B7/CD28 costimulatory interaction(Parra et al., J. Immunol., 158:637-642, 1997; Fenton et al., J.Immunother., 21:95-108, 1998).

Lymphocyte function associated antigen-1 (LFA-1) is expressed onleukocytes and interacts with ICAM-1 expressed on APCs and some tumorcells. This interaction induces T cell IL-2 and IFN-gamma production andcan thus complement but not substitute, the B7/CD28 costimulatoryinteraction (Fenton et al., 1998). LFA-1 is thus a further example of acostimulatory molecule that could be provided in a vaccination protocolin the various ways discussed above for B7.

Complete CTL activation and effector function requires Th cell helpthrough the interaction between the Th cell CD40L (CD40 ligand) moleculeand the CD40 molecule expressed by DCs (Ridge et al., Nature 393:474,1998; Bennett et al., Nature 393:478, 1998; Schoenberger et al., Nature393:480, 1998). This mechanism of this costimulatory signal is likely toinvolve upregulation of B7 and associated IL-6/IL-12 production by theDC (APC). The CD40-CD40L interaction thus complements the signal 1(antigen/MHC-TCR) and signal 2 (B7-CD28) interactions.

The use of anti-CD40 antibodies to stimulate DC cells directly, would beexpected to enhance a response to tumor associated antigens which arenormally encountered outside of an inflammatory context or are presentedby non-professional APCs (tumor cells). Other methods for inducingmaturation of dendritic cells, e.g., by increasing CD40-CD40Linteraction, or by contacting DCs with CpG-containingoligodeoxynucleotides or stimulatory sugar moieties from extracellularmatrix, are known in the art. In these situations Th help and B7costimulation signals are not provided. This mechanism might be used inthe context of antigen pulsed DC based therapies or in situations whereTh epitopes have not been defined within known tumor associated antigenprecursors.

When administered, the therapeutic compositions of the present inventionare administered in pharmaceutically acceptable preparations. Suchpreparations may routinely contain pharmaceutically acceptableconcentrations of salt, buffering agents, preservatives, compatiblecarriers, supplementary immune potentiating agents such as adjuvants andcytokines and optionally other therapeutic agents.

The preparations of the invention are administered in effective amounts.An effective amount is that amount of a pharmaceutical preparation thatalone, or together with further doses, stimulates the desired response.In the case of treating cancer, the desired response is inhibiting theprogression of the cancer. This may involve only slowing the progressionof the disease temporarily, although more preferably, it involveshalting the progression of the disease permanently. In the case ofinducing an immune response, the desired response is an increase inantibodies or T lymphocytes which are specific for the MAGE immunogen(s)employed. These desired responses can be monitored by routine methods orcan be monitored according to diagnostic methods of the inventiondiscussed herein.

Where it is desired to stimulate an immune response using a therapeuticcomposition of the invention, this may involve the stimulation of ahumoral antibody response resulting in an increase in antibody titer inserum, a clonal expansion of cytotoxic lymphocytes, or some otherdesirable immunologic response. It is believed that doses of immunogensranging from one nanogram/kilogram to 100 milligrams/kilogram, dependingupon the mode of administration, would be effective. The preferred rangeis believed to be between 500 nanograms and 500 micrograms per kilogram.The absolute amount will depend upon a variety of factors, including thematerial selected for administration, whether the administration is insingle or multiple doses, and individual patient parameters includingage, physical condition, size, weight, and the stage of the disease.These factors are well known to those of ordinary skill in the art andcan be addressed with no more than routine experimentation.

EXAMPLES Example 1 Identification of MAGE-A3 Epitopes Presented byHLA-DR1

Antigens encoded by MAGE-A3 and recognized by T cells are interestingtargets for tumor immunotherapy because they are strictly tumor-specificand shared by many tumors of various histological types. A number ofMAGE-A3 antigenic peptides presented by HLA class I molecules have beenused in clinical trials and regressions of melanoma metastasis have beenobserved. We report here the identification of additional MAGE-A3epitopes, including ACYEFLWGPRALVETS (MAGE-A3₂₆₇₋₂₈₂; SEQ ID NO:4) andGSDPACYEFLWGPRAL (MAGE-A3₂₆₃₋₂₇₈; SEQ ID NO:3), presented to CD4⁺ Tlymphocytes by HLA-DR1 molecules, which are expressed in approximately18% of Caucasians and 6% of Orientals. These new epitopes may be usefulboth for therapeutic vaccination and for the evaluation of the immuneresponse in cancer patients. To identify the epitopes, monocyte-deriveddendritic cells from a cancer patient were loaded with a recombinantMAGE-A3 protein and used to stimulate autologous CD4⁺ T cells. Thispatient had melanoma metastases expressing MAGE-A3 and was injected witha recombinant MAGE-A3 protein.

Materials and Methods

Cell lines, media, and reagents. The Epstein Barr Virus-transformed B(EBV-B) cell lines and the tumor cell lines MZ2-MEL43, NA41-MEL andSK37-MEL were cultured in IMDM (GIBCO BRL, Gaithersburg, Md.)supplemented with 10% fetal calf serum (GIBCO BRL), 0.24 mML-asparagine, 0.55 mM L-arginine, 1.5 mM L-glutamine (AAG), 100 U/mlpenicillin and 100 μg/ml streptomycin. Human recombinant IL-2 waspurchased from Eurocetus (Amsterdam, The Netherlands), IL-7 from Genzyme(Cambridge, Mass.), GM-CSF from Schering Plough (Brinny, England), TNF-αfrom R&D Systems (Abingdon, United Kingdom). Human recombinant IL-4,IL-6, and IL-12 were produced in our laboratory.

The PhoenixAMPHO cell line (kindly provided by Dr. Nolan, StanfordUniversity School of Medicine, Calif., USA) is a high titer amphotropicretrovirus producing cell line that has been generated by stabletransfection of 293T cells with a Moloney GagPol-IRES-Lyt 2 constructwith an RSV promoter and a pPGK hygro selectable marker. These cellswere then stably transfected with the Moloney amphotropic envelope genedriven by a CMV promoter and co-selected with the diphtheria toxinresistance gene (pHED-7). This producer cell line is helper virus free.

PhoenixAMPHO cells were cultured and passaged in 175 cm² flasks in DMEM(Life Technologies, Ghent, Belgium) supplemented with 10% heatinactivated FCS, 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/mlstreptomycin.

MAGE-A3 proteins. Two different MAGE-A3 proteins were used. One wasproduced in our laboratory in Spodoptera frugiperda (Sf9) insect cells,hereafter referred to as protein MAGE-A3^(insect). A baculovirusexpression system from PharMingen (San Diego, Calif.) was used. Thecoding sequence of MAGE-A3 was cloned in pAcGP67-A (PharMingen), abaculovirus transfer vector, downstream of the signal sequence of thegp67 surface protein of Autographa californica nuclear polyhedrosisvirus (AcNPV), a strain of baculovirus. For easier purification, asequence encoding a histidine tail was added at the C-terminus of thesequence of MAGE-A3. DNA of the recombinant plasmid was co-transfectedwith DNA of lethally mutated AcNPV into Sf9 insect cells.Co-transfection allows recombination between homologous regions of theplasmid and the virus, transferring the foreign gene from the vector tothe AcNPV DNA.

Purification of the protein contained in the supernatant of Sf9 insectcell cultures involved the following steps: anion exchange onDEAE-sephadex A-50 (Amersham-Pharmacia Biotech AB, Uppsala, Sweden),retention on HighTrap chelating column (Amersham-Pharmacia Biotech AB,Uppsala, Sweden), retention on High Trap chelating column(Amersham-Pharmacia Biotech AB, Uppsala, Sweden) saturated with NiCl₂,affinity chromatography with immobilized antibody 57B (kindly providedby Dr. G. Spagnoli, Department of Surgery, Basel, Switzerland),concentration and dialysis. The purification of the MAGE-A3 protein wasmonitored using a particle counting immunoassay, where the latex beadswere coated with purified F(ab′)₂ obtained from a goat immunized againstMAGE-A3.

The other MAGE-A3 protein, hereafter referred to as proteinMAGE-A3^(bacteria), was produced in Escherichia coli bySmithKline-Beecham Biologicals (Rixensart, Belgium). MAGE-A3^(bacteria)was produced as a recombinant His-MAGE-A3 protein (MAGE-A3 with a Histag) or as a recombinant LipoD-MAGE-A3-His protein. LipoD-MAGE-A3-Hiscontains one third of the lipidic form of the Haemophilus influenzaeprotein at its N-terminal residue and a polyhistidine marker at itsC-terminal residue. The proteins were purified by standardchromatographic procedures.

Construction of the retroviris encoding Ii-MA GE-A3. A recombinantretrovirus, pMFG-Ii80-MAGE-A3-(IRES)-ΔLNGFr was constructed(retro-Ii-MAGE). The sequence encoding a truncated form of the human lowaffinity nerve growth factor receptor (ΔLNGFr) was amplified fromplasmid pUC19-ΔLNGFr that was kindly provided by Dr. C. Traversari(Instituto Scientifico H. S. Raffaele, Milan, Italy). The PCRamplification was carried out using the following primers:

(SEQ ID NO:17) sense: 5′-CCCTCATGAGGGCAGGTGCCACCG-3′ (SEQ ID NO:18)antisense: 5′-CCCAGATCTCTAGAGGATTCCCCTGTTCCAC-3′This PCR introduces a BspH1 site at the start codon and a Bg/2 sitedownstream from the stop codon. A BamH1 site near the 3′ end wasdeleted. The PCR product was cloned in pCR2.1 and sequenced. The ΔLNGFrgene fragment was isolated from this vector as a BspH1-Not1 (from thepCR2.1 polylinker) fragment.

The IRES sequence derived from the encephalomyocarditis virus wastransferred from pGEM-EMC2 (kindly provided by Dr. J.-C. Renauld,Catholic University of Louvain, Brussels, Belgium) into pBluescript. AnEcoR1-Nco1 fragment from pBluescript-IRES was used for furtherconstruction.

Both the ΔNGFr and the IRES DNA fragments were ligated together intopCR2.1 EcoR1-Not1. This three fragment ligation resulted in a plasmidnamed pCR2.1-IRES-ΔLNGFr.

The IRES-ΔLNGFr sequence was then transferred into pMFG-Ii80, whichencodes the first 80 amino acids of the human invariant chain (Ii80). Acomplete MAGE-A3 cDNA was then ligated downstream of Ii80 intopMFG-Ii80-(IRES)-ΔLNGFr to form pMFG-Ii80-MAGE-A3-(IRES)-ΔLNGFr,allowing the simultaneous expression of the Ii-MAGE-A3 fusion proteinand the ΔLNGF receptor. The procedure for transducing cell lines hasbeen described previously.

High titer MAGEA3-encoding recombinant retrovirus stocks were generatedby introducing plasmid pMFG-Ii80-MAGE-A3-(IRES)-ΔLNGFr into PhoenixAMPHOpackaging cells by transfection, as described below. Retrovirus stockswere harvested and used for transduction as described below.

Generation of high titer MAGE-A3 encoding recombinant retrovirus. TheMAGE-A3 encoding retroviral vector plasmidpMFG-Ii80-MAGE-A3-(IRES)-ΔLNGFr was introduced into the PhoenixAMPHOpackaging cells by transfection. The MFG retroviral vector is derivedfrom Moloney murine leukemia virus and is lacking in a drug resistancemarker nor does it express any other potential antigenic protein exceptfor the inserted cDNA (Rivière, Proc. Natl. Acad. Sci. USA 92:6733-6737,1995). The transfection procedure is a modification of the calciumphosphate-mediated transfection protocol of Graham and van der Eb(Virology 54:536-539).

Twenty four hours prior to transfection, 10.8×10⁶ PhoenixAMPHO cellswere plated in 14 ml cell growth medium in a 75 cm² tissue culture flask(Falcon). After adding the cells, the flask was gently shaken forwardand backward to distribute cells evenly about the flask bottom. Thecells were incubated at 37° C. and 5% CO₂. At the time of transfection,when the cells should have reached a confluence of 70-80%, the mediumwas removed and was replaced by 14 ml fresh PhoenixAMPHO cell growthmedium containing 25 mM chloroquine (Sigma Chemical Co., St. Louis, Mo.,USA). A transfection cocktail was prepared in a 50 ml tube by adding 40μg retroviral vector plasmid DNA to water and diluting to 1575 μl finalvolume. To this DNA solution 225 μl of 2 M CaCl₂ (Sigma) was added.Then, 1800 μl of 2×HeBS (50 mM HEPES, 10 mM KCl, 12 mM dextrose, 280 mMNaCl and 1.5 mM Na₂HPO₄ dissolved in distilled water, filtered through0.2μ filter and stored at −20° C.) was added dropwise to the DNA/CaCl₂solution by vigorously bubbling for 15 seconds with an automaticpipette. The DNA/CaCl₂/HeBS mix was added immediately and dropwise ontothe cells and the flask was gently swirled to ensure uniform mixing ofDNA/CaPO₄ particles. The cells were incubated at 37° C./5% CO₂ for 7 to9 hours and the chloroquine containing medium was changed for freshPhoenixAMPHO cell growth medium. Approximately 24 hours prior to theharvest of the retroviral supernatant, the PhoenixAMPHO medium wasremoved and gently replaced by 9 ml of EBV cell growth medium (Iscove's)containing only 2.5% FCS. The retroviral supernatant was harvested 48hours following transfection by removing the medium from the cells andfiltering through a 0.45μ filter to remove cell debris. After harvestand filtration, the virus containing medium was kept on ice, aliquotedin appropriate volumes in 15 ml polypropylene tubes and stored at −80°C.

Retroviral transduction of EBV cell lines. The EBV transformed cellswere infected by resuspending the cells in an infection cocktail andcentrifugation. Target cells were resuspended in 60 mm tissue cultureplates (Falcon) at a density of 1.0×10⁶ cells in 4 ml infectioncocktail. The plates were centrifuged for 2 hours at 32° C. and 1200 rcfin an IEC centrifuge, rotor type 228. For each plate to be transduced, 4ml of injection cocktail was prepared by diluting the viral supernatant1:2 in EBV cell growth medium and adding protamine sulfate to a finalconcentration of 6 μg/ml. Centrifugation was followed by another 2 hoursof incubation in a humidified incubator at 37° C. and cells weretransferred to 4 ml of target cell growth medium. This transductioncycle was carried out immediately after plating the cells and wasrepeated at 24 and 48 hours.

Dendritic cells and CD4⁺ responder T cells. Blood cells were collectedas buffy-coat preparations from melanoma patient DDHK2, and processedessentially as described previously in PCT/US99/21230. Briefly,peripheral blood mononuclear cells (PBMC) were isolated bycentrifugation on Lymphoprep (Nycomed Pharma, Oslo, Norway). In order tominimize contamination of PBMC by platelets, the preparation was firstcentrifuged for 20 min/1000 rpm at room temperature. After removal ofthe top 20-25 ml, containing most of the platelets, the tubes werecentrifuged for 20 min/1500 rpm at room temperature. PBMC were depletedof T cells by rosetting with 2-aminoethylisothiouronium (Sigma) treatedsheep erythrocytes. The lymphocyte-depleted PBMC were left to adhere for2 hours at 37° C. in culture flasks (Falcon) at a density of 2×10⁶cells/ml in RPMI 1640 medium supplemented with L-asparagine (0.24 mM),L-arginine (0.55 mM), L-glutamine (1.5 mM) and 1% autologous serum(complete medium). Non-adherent cells were discarded.

Adherent cells (dendritic cells) were obtained by culturing monocytes inthe presence of IL-4 (200 U/ml) and GM-CSF (70 ng/ml) in RPMI 1640medium supplemented with AAG and 1% autologous plasma. One fourth of themedium was replaced by fresh medium and cytokines every two days. On day7, the non-adherent cell population was used as a source of enricheddendritic cells. Rosetted T cells were treated with NH₄Cl (160 mM) tolyse the sheep erythrocytes, and washed. CD4⁺ T lymphocytes wereisolated from rosetted T cells by positive selection using an anti-CD4monoclonal antibody coupled to magnetic microbeads (Miltenyi Biotech,Germany) and by sorting through a MACS, as recommended by themanufacturer.

Mixed lymphocytes/dendritic cells culture. Dendritic cells (5×10⁵) wereincubated at 37° C., 5% CO₂, for 20 h in complete RPMI mediumsupplemented with IL-4, GM-CSF and TNF-α (5 ng/ml) in the presence ofMAGE-A3^(bacteria) (20 μg/ml). Cells were washed and added at 10⁴ perround-bottomed microwell to 10⁵ CD4⁺ T lymphocytes in 200 μl IMDMsupplemented with AAG and 1% autologous plasma in the presence of IL-6(1,000 U/ml) and IL-12 (10 ng/ml). The CD4⁺ lymphocytes wererestimulated on days 7, 14, 21 and 28 with autologous dendritic cellsfreshly loaded with MAGE-A3^(bacteria) and grown in IMDM supplementedwith AAG and 1% autologous plasma (hereafter referred to as completeIMDM) supplemented with IL-2 (10 U/ml) and IL-7 (5 ng/ml). Aliquots ofeach microculture (˜5,000 cells) were assessed on days 35 and 42 fortheir capacity to produce IFN-γ when stimulated with ˜20,000 autologousEBV-B cells loaded for 20 h with either 20 μg/ml of MAGE-A3^(bacteria),MAGE-A3^(insect), or ovalbumin. After 20 h of co-culture in round-bottommicrowells and in 100 μl complete IMDM medium supplemented with IL-2 (25U/ml), IFN-γ released in the supernatant was measured by ELISA usingreagents from Medgenix Diagnostics-Biosource (Fleurus, Belgium).

CD4⁺ T cell clones. Cells from positive microculture F4 that were clonedby limiting dilution, using irradiated autologous EBV-B cells transducedwith retro-Ii.MAGE-A3 (5×10³-2×10⁴ cells) as stimulator cells.Irradiated allogeneic LG2-EBV cells (5×10³-10⁴) were used as feedercells. CD4⁺ T cell clones were supplemented once a week with freshculture medium in the presence of IL-2 (50 U/ml), IL-7 (5 ng/ml) andIL-4 (5 U/ml).

Recognition assays with peptides. Peptides were synthesized on solidphase using F-moc for transient NH2-terminal protection and werecharacterized using mass spectrometry. All peptides were >90% pure, asindicated by analytical HPLC. Lyophilized peptides were dissolved at 5mg/ml in 10 mM acetic acid and 10% DMSO, and stored at −20° C. EBV-Bcells were distributed at 20,000 cells per round-bottomed microwell andincubated for 2 h at 37° C. in the presence of the different peptides,the indicated concentrations representing their concentrations duringthe incubation step. CD4⁺ T lymphocytes (5,000) were added in 100 μl ofcomplete IMDM (GIBCO) medium supplemented with IL-2 (25 U/ml).Supernatants were harvested after 20 h of co-culture and IFN-γproduction was measured by ELISA.

Recognition of tumor cells. Tumor cells were distributed at 20,000 cellsper round-bottomed microwell together with 5,000 CD4⁺ T lymphocytes in100 μl of complete IMDM medium in the presence of IL-2 (25 U/ml).Supernatants were harvested after 20 h of co-culture and the IFN-γproduction was measured by ELISA.

Results

To identify new MAGE-A3 epitopes presented by HLA class II molecules,monocyte-derived dendritic cells (DC) from melanoma patient DDHK2 werecultured in autologous plasma and incubated overnight with a recombinantMAGE-A3 protein and with TNF-α to induce their maturation. These cellswere then used to stimulate autologous CD4⁺ T lymphocytes. In previousexperiments, a large number of the CD4⁺ T cell clones obtained with thesame method were apparently directed against bacterial contaminants inthe batch of protein. Therefore, a MAGE-A3 protein produced inEscherichia coli (MAGE-A3^(bacteria)) was used to stimulate thelymphocytes, and a MAGE-A3 protein produced in Spocloptera frugiperdainsect cells (MAGE-A3^(insect)) to test the specificity of the responderlymphoctyes (data not shown).

A CD4⁺ T cell clone directed against a MAGE-A3 antigen. A total of 96microcultures were set up, each containing 10⁵ CD4⁺ cells and 10⁴autologous stimulator DC loaded with protein MAGE-A3^(bacteria) asstimulator cells. Responder cells were restimulated three times atweekly intervals with DC loaded with the protein. After a resting periodof two weeks, responder cells of each microculture were tested on days35 and 42 for IFN-γ production after stimulation with autologous EBV-Bcells loaded with either MAGE-A3^(insect), MAGE-A3^(bacteria) orovalbumin. Twelve microcultures specifically produced a high level ofIFN-γ after stimulation with protein MAGE-A3^(insect) and proteinMAGE-A3^(bacteria). One of them, F4, was cloned by limiting dilutionusing autologous EBV-B cells transduced with retro-Ii.MAGE-A3 asstimulator cells. Several positive clones were obtained, including CD4⁺T cell clone MAGJ569/F4.3. This clone recognized autologous EBV-B cellsloaded with either protein MAGE-A3^(insect) or proteinMAGE-A3^(bacteria), or EBV-B cells transduced with retro-Ii.MAGE-A3.

Autologous DDHK2-EBV-B cells were pulsed for 20 h with 20 μg/ml ofprotein MAGE-A3^(insect) or protein MAGE-A3^(bacteria). Protein-pulsedEBV-B cells (20,000) or EBV-B cells transduced with a retrovirusencoding a fusion protein composed of MAGE-A3 and a truncated humaninvariant chain (retro-Ii.MAGE-A3) were incubated for 20 h in microwellswith CD4⁺ clone MAGJ569/F4.3 cells (5,000). IFN-γ production wasmeasured by ELISA (FIG. 1). The results shown represent the average oftriplicate cultures.

Clone MA GJ569/F4.3 recognized peptide ACYEFLWGPRALVETS (SEQ ID NO:4). Aset of peptides of 16 amino acids, which overlapped by 12 and coveredthe entire MAGE-A3 protein sequence, was screened: autologous EBV-Bcells were pulsed with each of these peptides and tested for recognitionby clone MAGJ569/F4.3. It produced IFN-γ after stimulation with twooverlapping peptides, namely MAGE-A3₂₆₃₋₂₇₈ (GSDPACYEFLWGPRAL; SEQ IDNO:3) and MAGE-A3₂₆₇₋₂₈₂ (ACYEFLWGPRALVETS; SEQ ID NO:4).

DDHK EBV-B cells (20,000) were incubated in microwells for 2 hours withdifferent concentrations of the MAGE-A3 peptides. IFN-γ production wasmeasured by ELISA after hours of coculture with CD4⁺ clone MAGJ569/F4.3(5,000 cells) (FIG. 2). The experiments were performed twice.

FIG. 2A shows Experiment I. A number of MAGE-A3 peptides of differentlengths were tested and recognized by clone MAGJ569/F4.3. The peptidestested were GSDPACYEFLWGPRAL (MAGE-A3₂₆₃₋₂₇₈; SEQ ID NO:3),ACYEFLWGPRALVETS (MAGE-A3₂₆₇₋₂₈₂; SEQ ID NO:4), ACYEFLWGPRALVE (SEQ IDNO:7), ACYEFLWGPRALV (SEQ ID NO:8), ACYEFLWGPRAL (SEQ ID NO:9),ACYEFLWGPRA (SEQ ID NO:10), ACYEFLWGPR (SEQ ID NO:11), CYEFLWGPRALVE(SEQ ID NO:12), YEFLWGPRALVE (SEQ ID NO:13), and EFLWGPRALVE (SEQ IDNO:14). Of these, GSDPACYEFLWGPRAL (SEQ ID NO:3), ACYEFLWGPRALVETS (SEQID NO:4), and ACYEFLWGPRALVE (SEQ ID NO:7) were well recognized. It isexpected that ACYEFLWGPRALVET (SEQ ID NO:16), as intermediate betweenthe amino acid sequences of SEQ ID NO:4 and SEQ ID NO:7, also would bewell recognized.

FIG. 2B shows Experiment II. For this second experiment, concentrationof peptides were measured by optical density. The peptides tested wereGSDPACYEFLWGPRAL (SEQ ID NO:3), ACYEFLWGPRALVETS (SEQ ID NO:4),ACYEFLWGPRALVE (SEQ ID NO:7) and ACYEFLWGPRALV (SEQ ID NO:8). Of these,GSDPACYEFLWGPRAL (SEQ ID NO:3), ACYEFLWGPRALVETS (SEQ ID NO:4), andACYEFLWGPRALVE (SEQ ID NO:7) were well recognized, while ACYEFLWGPRALV(SEQ ID NO:8) was recognized but only at higher concentrations ofpeptide.

The peptide is presented by HLA-DR1 molecules. DDHK2-EBV transduced withretro-Ii.MAGE-A3 (20,000) were cocultured for 20 hours with CD4⁺ cloneMAGJ569/F4.3 cells (5,000), in the presence of either monoclonalantibody B7.21 (anti-DP), SPV L3 (anti-DQ), or AH89 (anti-DR). IFN-γproduction was measured by ELISA. The recognition by clone MAGJ569/F4.3of autologous EBV-B cells loaded with peptide MAGE-A3₂₆₇₋₂₈₂(ACYEFLWGPRALVETS; SEQ ID NO:4) was abolished by an anti-HLA-DRantibody, but not by antibodies against HLA-DP or HLA-DQ (FIG. 3).

Melanoma patient DDHK2 was typed HLA-DR1, DR15 and DR51. PeptideACYEFLWGPRALVETS (SEQ ID NO:4) was loaded on several EBV-B cell linesexpressing DR1, DR15 or DR51. All and only those expressing DR1 wereable to present the peptide to clone MAGJ569/F4.3 (Table 1). AutologousDDHK2-EBV and allogeneic EBV-B cells (20,000) were incubated for 1 hourwith 5 μg/ml of peptide ACYEFLWGPRALVETS (MAGE-A3₂₆₇₋₂₈₂; SEQ ID NO:4)and washed. Peptide-pulsed EBV-B cells (20,000) were then incubated withCD4⁺ clone MAGJ565/F4.3 cells (5,000). IFN-γ production was measured byELISA after 20 hours of co-culture.

TABLE 1 Presentation of the MAGE-A3 peptide (MAGE362) by HLA-DR1 cellsEBV-B cell line Serological specificity IFN-γ production (pg/ml) DR1positive DDHK2 DR1 DR15 DR51 >4000 LB1158 DR1 DR13 DR52 2439 LB831 DR1DR7 DR53 2037 LB2138 DR1 DR13 1810 DR1 negative LB650 DR7 DR15 DR51 DR5383 LB1870 DR15 DR53 DR7 88 LB1856 DR15 49 LB2095 DR13 DR15 DR51 DR52 22

Recognition of tumor cell lines. Three DR1 melanoma cell linesexpressing MAGE-A3 were tested for their ability to stimulate CD4⁺ Tcell clone MAGJ569/F4.3 to produce IFN-γ. Tumor cells were pretreatedfor 48 h with 100 U/ml of IFN-γ and were pulsed in microwells (20,000)for 1 h with 1 μg/ml of peptide ACYEFLWGPRALVETS (MAGE-A3₂₆₇₋₂₈₂; SEQ IDNO:4). IFN-γ production was measured by ELISA after 20 h of coculturewith CD4⁺ clone MAGJ610/F4.3 (5,000). Autologous EBV-stimulator cellstransduced with a retrovirus encoding Ii.MAGE-A-3 were used as positivecontrols.

One of the melanoma cell lines, NA41-MEL, slightly stimulated the CD4⁺clone MAGJ569/F4.3 to produce IFN-γ (FIG. 4). Treatment of this cellline with IFN-γ improved its recognition.

Example 2 Cloning of T Cell Receptors and Recognition of Homologous MAGEPeptides

Materials and Methods

Cell lines, media, and reagents. The Epstein Barr Virus-transformed B(EBV-B) cell lines and the tumor cell lines were cultured in IMDM (GibcoBRL, Gaithersburg, Md., USA) supplemented with 10% fetal calf serum(Gibco BRL), 0.24 mM L-asparagine, 0.55 mM L-arginine, 1.5 mML-glutamine (AAG), 100 U/ml penicillin and 100 μg/ml streptomycin. Humanrecombinant IL-2 was purchased from Eurocetus (Amsterdam, TheNetherlands), IL-7 from Genzyme (Cambridge, Mass., USA), GM-CSF fromSchering Plough (Brinny, Ireland), TNF-α from R&D Systems (Abingdon,United Kingdom). Human recombinant IL-4, IL-6, and IL-12 were producedin our laboratory.

MAGE-3 proteins. Two different MAGE-3 proteins were used. One wasproduced in our laboratory in Spodoptera frugiperda (Sf9) insect cellsusing a baculovirus expression system (PharMingen, San Diego, Calif.,USA), as described previously (Schultz et al., Cancer Res. 60:6272,2000). It will be referred to hereafter as protein MAGE-3^(insect). Theother MAGE-3 protein, hereafter referred to as proteinMAGE-3^(bacteria), was produced in Escherichia coli by GlaxoSmithKlineBiologicals (Rixensart, Belgium), as previously reported (Chaux et al.,J. Exp. Med. 189:767, 1999). It contains a sequence of several histidineresidues at the N-terminus of the protein. The recombinant MAGE-3protein used in the vaccine is a fusion protein with a lipidated proteinD derived from H. influenzae at its N-terminus, and a sequence ofseveral histidine residues at the C-terminus of the protein (Prot.DMAGE-3/His). The inclusion of the first 109 residues of the protein D asa fusion partner Was expected to improve the immunogenicity and toprovide the vaccine protein with additional bystander help properties,whereas the inclusion of a His affinity tail facilitated thepurification of the fusion protein. The protein was produced in E. coliand extensively purified to eliminate bacterial contaminants.

Construction of the retrovirus encoding MAGE-A3, MAGE-A1, MAGE-A4 andIi-MAGE-A3. The retroviral vector encoding MAGE-A1, -A3 and -A4 werederived from the LXSN backbone, an expression vector derived fromMoloney murine leukemia virus (Clontech, Palo Alto, Calif., USA). Theyrespectively encode the full-length MAGE-A1, -A3 and -A4 proteins underthe control of the long terminal repeat (LTR), and the truncated form ofthe human low affinity nerve growth factor receptor (LNGFR) driven bythe SV40 promoter. For transduction, EBV-B cell lines were co-cultivatedwith irradiated Am12 vector-producing cells (Miller and Rosman,Biotechniques 7:980, 1989) in the presence of polybrene (0.8 mg/ml) for72 h. A pure population of transduced cells was obtained byimmunoselection with anti-LNGFR mAb 20.4 (ATCC, Rockville, Md., USA) andgoat anti-mouse IgG FITC (Becton Dickinson Immunocytometry Systems, SanJose, Calif., USA). For producing the retrovirus encoding Ii-MAGE-3, thesequence encoding a truncated form of LNGFR was amplified from plasmidpUC19-ΔLNGFR that was kindly provided by Dr C. Traversari (IstitutoScientifico H.S. Raffaele, Milano, Italy). Briefly, LNGFR was ligatedinto pCR2.1 to an IRES sequence, derived from the encephalomyocarditisvirus. The IRES-ΔLNGFR sequence was then transferred into pMFG-Ii80,which encodes the first 80 amino acids of the human invariant chain(Ii80). A complete MAGE-3 cDNA was then ligated downstream Ii80 intopMFG-Ii80-IRES-ΔLNGFR, allowing the simultaneous expression of theIi-MAGE-3 fusion protein and the truncated LNGFR receptor. The procedurefor transducing cell lines has been described previously (Mavilio etal., Blood 83:1988, 1994).

Dendritic cells and CD4⁺ responder T cells. Blood cells were collectedas buffy-coat preparations from melanoma patient DDHK2, and processed asdescribed above and previously (Schultz et al., J. Exp. Med. 195:391,2002). Briefly, dendritic cells were obtained by culturing monocytes inthe presence of IL-4 (200 U/ml) and GM-CSF (70 ng/ml) in RPMI 1640medium supplemented with AAG and 1% autologous plasma. One fourth of themedium was replaced by fresh medium and cytokines every two days. On day7, the non-adherent cell population was used as a source of enricheddendritic cells. Rosetted T cells were treated with NH₄Cl (160 mM) tolyse the sheep erythrocytes, and washed. CD4⁺ T lymphocytes wereisolated from rosetted T cells by positive selection using magneticmicrobeads coupled to an anti-CD4 monoclonal antibody (Miltenyi Biotech,Germany) and by sorting through a MACS, as recommended by themanufacturer (Miltenyi Biotech).

Mixed lymphocytes/dendritic cells culture. Dendritic cells (5×10⁵/ml)were incubated at 37° C., 5% CO₂, for 20 h in complete RPMI mediumsupplemented with IL-4, GM-CSF and TNF-α (1 ng/ml) in the presence ofMAGE-3^(bacteria) (20 μg/ml). The small amount of TNF does not inducethe maturation of DC, as measured by CD83 expression. However, we cannotexclude that additional TNF produced by T cells or activation throughCD40-CD40L during the co-culture led to DC maturation. Cells were washedand added at 10⁴ per round-bottomed microwell to 10⁵ autologous CD4⁺ Tlymphocytes in 200 μl IMDM supplemented with AAG and 1% autologousplasma in the presence of IL-6 (1,000 U/ml) and IL-12 (10 ng/ml). TheCD4⁺ T lymphocytes were restimulated on days 7, 14, 21 and 28 withautologous dendritic cells freshly loaded with MAGE-3^(bacteria) andgrown in IMDM supplemented with AAG and 1% autologous plasma (hereafterreferred to as complete IMDM) supplemented with IL-2 (10 U/ml) and IL-7(5 ng/ml). Aliquots of each microculture (˜5,000 cells) were assessed onday 42 for their capacity to produce IFN-γ when stimulated with ˜20,000autologous EBV-B cells loaded for 20 h with either 20 μg/ml ofMAGE-3^(bacteria), MAGE-3^(insect), or ovalbumin. After 20 h ofco-culture in round-bottomed microwells and in 100 μl complete IMDMmedium supplemented with IL-2 (25 U/ml), IFN-γ released in thesupernatant was measured by ELISA using reagents from MedgenixDiagnostics-Biosource (Fleurus, Belgium).

CD4⁺ T cell clones. Cells from positive microcultures were cloned bylimiting dilution, using irradiated autologous EBV-B cells transducedwith retro-Ii.MAGE-3 (5×10³-2×10⁴ cells) as stimulator cells. Irradiatedallogeneic LG2-EBV cells (5×10³-104) were used as feeder cells. CD4⁺ Tcell clones were supplemented once a week with fresh culture medium inthe presence of IL-2 (50 U/ml), IL-7 (5 ng/ml) and IL-4 (5 U/ml).

TCR analysis. For TCR analysis, 3×10⁵ cells from each clone were usedfor extracting RNA with the Tripure reagent (Boehringer Mannheim,Mannheim, Germany) and converted to cDNA at 42° C. for 90 min with 200 UM-murine leukemia virus reverse transcriptase (Life Technologies,Merelbeke, Belgium). TCR Vα and Vβ usage was assessed by PCRamplification by using a complete panel of Vα- or Vβ-specific senseprimers and Cα or Cβ antisense primer, respectively (Genevée et al.,Eur. J. Immunol. 22:1261, 1992). Primers were chosen on the basis ofdescribed panels of TCR V region oligonucleotides, and with alignmentsof TCR sequences available at from IMGT, the internationalImMunoGeneTics information system®, or EMBL-EBI (European BioinformaticsInstitute). Each PCR product was purified and sequenced to obtain acomplete identification of the CDR3 region.

Recognition assays with peptides. Peptides were synthesized on solidphase using Fmoc for transient NH₂-terminal protection and werecharacterized using mass spectrometry. All peptides were >90% pure, asindicated by analytical HPLC. Lyophilized peptides were dissolved at 5mg/ml in 10 mM acetic acid and 10% DMSO, and stored at −20° C. EBV-Bcells were distributed at 20,000 cells per round-bottomed microwell andincubated for 2 h at 37° C. in the presence of the different peptides,the indicated concentrations representing their concentrations duringthe incubation step. CD4⁺ T lymphocytes (5,000) were added in 100 μl ofcomplete IMDM (Gibco BRL) medium supplemented with IL-2 (25 U/ml).Supernatants were harvested after 20 h of co-culture and IFN-γproduction was measured by ELISA. The peptides used in FIG. 6 correspondto the MAGE-1₂₆₀₋₂₇₅, MAGE-2₂₆₇₋₂₈₂, MAGE-3₂₆₇₋₂₈₂, MAGE-4₂₆₈₋₂₈₃,MAGE-6₂₆₇₋₂₈₂, MAGE-10₂₉₂₋₃₀₇, MAGE-11₂₇₀₋₂₈₅, and MAGE-12₂₆₇₋₂₈₂protein sequences.

Recognition assays with cell lysates. EBV-B cells (5×10⁴) were lysed in50 μl of complete RPMI medium by three cycles of rapid freeze-thawing.HLA-DR1 monocyte-derived dendritic cells (2.5×10⁴) were then added tothe lysates in 150 μl of complete RPMI supplemented with IL-4 (100 U/ml)and GM-CSF (70 ng/ml) and kept at 37° C. for 24 h. Dendritic cells werewashed and 5,000 CD4⁺ lymphocytes were added in 150 μl of complete IMDMsupplemented with IL-2 (25 U/ml). Supernatants were harvested after 20 hof co-culture and IFN-γ production was measured by ELISA.

Recognition of tumor cells. Tumor cells were distributed at 20,000 cellsper round-bottomed microwell together with 5,000 CD4⁺ T lymphocytes in100 μl of complete IMDM medium in the presence of IL-2 (25 U/ml).Supernatants were harvested after 20 h of co-culture and IFN-γproduction was measured by ELISA. For measuring lytic activity, cellswere labeled with 100 μCi of Na(⁵¹Cr)O₄, and 1,000 targets were added tothe T cells at different effector-to-target ratios. Chromium release wasmeasured after 4 h of incubation at 37° C.

HLA-DR pepticle binding assay. Purification of HLA-DR molecules andpeptide binding assays were performed as previously described (Texier etal., J. Immunol. 164:3177, 2000; Texier et al., Eur. J. Immunol.31:1837). Briefly, HLA-DR molecules were purified from EBV-B homozygouscell lines by affinity chromatography. They were incubated withdifferent concentrations of competitor peptide and an appropriatebiotinylated peptide. The biotinylated peptides were the following: HA306-318 (PKYVKQNTLKLAT; SEQ ID NO:19) for DRB1*0101 (1 nM, pH 6),DRB1*0401 (30 nM, pH 6), DRB1*1101 (20 nM, pH 5) and DRB 5*0101 (5 nM,pH 5.5), YKL (AAYAAAKAAALAA; SEQ ID NO:20) for DRB1*0701 (10 nM, pH 5),A3 152-166 (EAEQLRAYLDGTGVE; SEQ ID NO:21) for DRB1*1501 (10 nM, pH4.5), MT 2-16 (AKTIAYDEEARRGLE; SEQ ID NO:22) for DRB1*0301 (100 nM, pH4.5), B1 21-36 (TERVRLVTRHIYNREE; SEQ ID NO:23) for DRB1*1301 (200 nM,pH 4.5) and LOL 191-210 (ESWGAVWRIDTPDKLTGPFT; SEQ ID NO:24) forDRB3*0101 (5 nM, pH 5.5). The binding was evaluated in a fluorescenceassay. Data were expressed as the peptide concentration that preventedbinding of 50% of the labeled peptide (IC₅₀). Average were deduced fromat least two independent experiments. Unlabeled forms of thebiotinylated peptides were used as reference peptides to assess thevalidity of each experiment.

Results

Derivation of Anti-MAGE-3 CD4⁺ T Cell Clones

Patient DDHK2 was vaccinated with recombinant protein Prot.D MAGE-3/HIS,which was produced in E. coli as a fusion protein with lipidated proteinD derived from H. influenzae at the N-terminus, and a sequence ofseveral histidine residues at the C-terminus of the MAGE-3 protein.Injections were given without adjuvant at 3-week intervals,intradernally and subcutaneously. Blood cells were collected two weeksafter the fourth injection of the MAGE-3 protein. Monocyte-deriveddendritic cells (DC) were loaded overnight with a MAGE-3 proteinproduced in bacteria (MAGE-3^(baceria)). Autologous plasma was used toavoid loading the DC with allogeneic proteins. In two independentexperiments, a total of 192 microcultures of 10⁵ CD4⁺ T cells and 10⁴stimulator cells were set up. To favor the activation of Th1lymphocytes, the culture medium was supplemented with IL-12. After fourweekly re-stimulations with protein-loaded DC, the responder cells weretested for their ability to secrete IFN-γ upon stimulation with theantigen. Considering that a large proportion of the CD4⁺ T cellsobtained in our initial experiments appeared to be directed againstbacterial contaminants, we used a protein produced in insect cells(MAGE-3^(insect)) for this test. Seven microcultures that specificallyproduced IFN-γ were cloned and restimulated with autologous EBV-B cellstransduced with a retroviral construct encoding a truncated humaninvariant chain (Ii) fused with the MAGE-3 protein (retro-Ii.MAGE-3)(Chaux et al., J. Exp. Med. 189:767, 1999; Sanderson et al., Proc. Natl.Acad. Sci. USA 92:7217, 1995). In this chimeric protein, signals withinthe invariant chain should target the MAGE-3 protein to the class IIantigen-processing compartments (Sanderson et al., 1995).

Anti-MAGE-3 CD4⁺ T cell clones were obtained from only three of theseven microcultures that were cloned. These clones (referred to in thisExample as clone 1, clone 2 and clone 3; clone 1 is the same cell cloneas clone MAGJ569/F4.3 of Example 1 above) recognized autologous EBV-Bcells (stimulator cells) either loaded with 20 μg/ml of proteinMAGE-3^(bacteria) or MAGE-3^(insect), or transduced withretro-Ii.MAGE-3. Stimulator cells (20,000) were co-cultured overnightwith 5,000 CD4⁺ T cells. The concentration of IFN-γ produced in themedium was measured by ELISA. The results shown (FIG. 5) represent theaverage of triplicate co-cultures.

The T cell receptors (TCRs) of the T cell clones were identified asdescribed above by PCR amplification using Vα- or Vβ-specific senseprimers and Cα or Cβ antisense primers, followed by sequencing of theproduct, resulting in obtaining the sequences of the CDR3 region of eachof the TCRs. Each of these three clones had a different TCR, as shown inTable 2.

TABLE 2 TCR sequences of the three anti-MAGE-3 CD4⁺ T cell clones CloneV α CHAIN TCRα clone 1 V9-2*01-J49*01 TCAGCGGTGTACTTC S  A  V  Y  Fclone 2 V36/DV7*04-J37*01 TCGGCCATCTACCTC S  A  I  Y  L clone 3V27*04-J52*01 ACAGGCCTCTACCTC T  G  L  Y  L β CHAIN TCRβ clone 1V28*01-J2-2*01 ACATCTATGTACCTC T  S  M  Y  L clone 2 V9*01-J2-1*01TCAGCTTTGTATTTC S  A  L  Y   F clone 3 V6-3*01-J1-2*01 ACATCTGTGTACTTCT  S  V  Y  F Clone CDR3 J α CHAIN clone 1TGTGCTCTTGAGAACACCGGTAACCAGTTCTATTTTGGG ACAGGGACAAGTTTGACGGTCATTC  A  L  E  N  T  G  N  Q  F  Y  F  G  T   G  T  S  L  T  V   I clone 2TGTGCTGTGGTGTCTGGCAACACAGGCAAACTAATCTTTGGG CAAGGGACAACTTTACAAGTAAAAC  A  V  V  S  G  N   T  G  K  L  I F  G Q  G  T  T  L  Q  G  I clone 3TGTGCAGGAAGGGGAAGAGGTACTAGCTATGGAAAGCTGACATTTGGACAAGGGACCATCTTGACTGTCCATC  A  G   R  G  R  G  T  S   Y G  K   L  T  F  G  Q  G  T  I   L  T  V  H β CHAIN clone 1TGTGCCAGCAGACCCTTTCCCGGGGAGCTGTTTTTTGGA GAAGGCTCTAGGCTGACCGTACTGC  A  S  R  P  F  P  G  E  L   F  F  G  E  G  S  R  L  T  V   L clone 2TGTGCCAGCAGCGTGTACTCCAATGAGCAGTTCTTCGGG CCAGGGACACGGCTCACCGTGCTAC  A  S  S   V  Y  S  N  E  Q  F   F  G  P   G  T  R  L  T  V   L clone3 TGTGCCAGCAGTCTGACAGGGACCAACTATGGCTACACCTTCGGT TCGGGGACCAGGTTAACCGTTGTA C  A  S  S  L  T  G  T  N   Y  G  Y  T  F   G    S  G  T  R  L  T   V GClone 1: Alpha: SEQ ID NO:45(tcagcggtgtacttctgtgctcttgagaacaccggtaaccagttctattttgggacagggacaagtttgacggtcatt) SEQ ID NO:46 (SAVYFCALENTGNQFYFGTGTSLTVI) CDR3α: SEQID NO:47 (CALENTGNQFYFGTG) Beta: SEQ ID NO:48(acatctatgtacctctgtgccagcagaccctttcccggggagctgttttttggagaaggctctaggctgaccgtactg) SEQ ID NO:49 (TSMYLCASRPFPGELFFGEGSRLTVL) CDR3β SEQ IDNO:50 (CASRPFPGELFFG) Clone 2: Alpha: SEQ ID NO:51(tcggccatctacctctgtgctgtggtgtctggcaacacaggcaaactaatctttgggcaagggacaactttacaagtaaaa) SEQ ID NO:52 (SAIYLCAVVSGNTGKLIFGQGTTLQGI) CDR3α:SEQ ID NO:53 (CAVVSGNTGKLIFG) Beta: SEQ ID NO:54(tcagctttgtatttctgtgccagcagcgtgtactccaatgagcagttcttcgggccagggacacggctcaccgtgcta) SEQ ID NO:55 (SALYFCASSVYSNEQFFGPGTRLTVL) CDR3β SEQ IDNO:56 (CASSVYSNEQFFG) Clone 3: alpha: SEQ ID NO:57(acaggcctctacctctgtgcaggaaggggaagaggtactagctatggaaagctgacatttggacaagggaccatcttgactgtccat) SEQ ID NO:58 (TGLYLCAGRGRGTSYGKLTFGQGTILTVH)CDR3α: SEQ ID NO:59 (CAGRGRGTSYGKLTFG) Beta: SEQ ID NO:60(acatctgtgtacttctgtgccagcagtctgacagggaccaactatggctacaccttcggttcggggaccaggttaaccgttgta) SEQ ID NO:61 (TSVYFCASSLTGTNYGYTFGSGTRLTVG)CDR3β: SEQ ID NO:62 (CASSLTGTNYGYTFG) ^(a)V and J rearrangements wereattributed according to the nomenclature available from IMGT, theinternational ImMunoGeneTics information system ®, or EMBL-EBI (EuropeanBioinformatics Institute).Identification of the Antigenic Peptides

We tested for recognition by each of the three clones autologous EBV-Bcells pulsed with a set of peptides of 16 amino acids, overlapping by 12residues and covering the complete MAGE-3 sequence. DDHK2 EBV-B cellswere distributed in microwells (2×10⁴ cells) and incubated for 2 h withthe indicated concentrations of peptides. 5×10³ cells from eachautologous CD4⁺ T cell clone were added and the presence of IFN-γ in thesupernatant was measured by ELISA after overnight co-culture. Theresults shown in FIG. 6 represent the average of triplicate co-cultures.

Two overlapping peptides, GSDPACYEFLWGPRAL (MAGE-3₂₆₃₋₂₇₈; SEQ ID NO:3)and ACYEFLWGPRALVETS (MAGE-3₂₆₇₋₂₈₂; SEQ ID NO:4), stimulated theproduction of IFN-γ by all three clones (FIG. 6). The latter peptide(MAGE-12₂₆₇₋₂₈₂) is also encoded by MAGE-12 (SEQ ID NO:43 is thenucleotide sequence, SEQ ID NO:44 is the amino acid sequence). Testingshorter peptides indicated that the most efficiently recognized peptidewas slightly different for each clone: ACYEFLWGPRALVETS (SEQ ID NO:4)for clone 1, ACYEFLWGPRALVE (SEQ ID NO:7) for clone 2, and bothACYEFLWGPRALVET (SEQ ID NO:16) and ACYEFLWGPRALVETS (SEQ ID NO:3) forclone 3 (FIG. 6).

The Antigenic Peptide is Presented to T Cells by HLA-DR1 Molecules

For each of the three clones, the recognition of autologous EBV-B cellsloaded with peptide MAGE-3₂₆₇₋₂₈₂ was abolished by an anti-HLA-DRantibody, but not by antibodies against HLA-DP or HLA-DQ (data notshown). Patient DDHK2 was typed HLA-DR1, DR15 and DR51. PeptideACYEFLWGPRALVETS (SEQ ID NO:4) was loaded on several EBV-B cell linessharing HLA-DR molecules with patient DDHK2. All and only thoseexpressing DR1 were recognized by the three clones when loaded with thepeptide (Table 3).

TABLE 3 MAGE-3 peptide ACYEFLWGPRALVETS is presented by HLA-DR1^(a)EBV-B IFN-γ production (pg/ml) cell line Serological specificity clone 1clone 2 clone 3 DR1 positive DDHK2 DR1 DR15 DR51 >4,000 >4,000 >4,000LB1158 DR1 DR13 DR52 2,439 2,029 1,698 LB831 DR1 DR7 DR53 2,037 1,4181,371 LB2138 DR1 DR13 1,810 2,046 2,053 DR1 negative LB650 DR7 DR15 DR51DR53 83 160 150 LB1870 DR15 DR53 DR7 88 104 119 LB1856 DR15 49 187 164LB2095 DR13 DR15 DR51 DR52 22 172 125 ^(a)EBV-B cells were incubated for2 h with 5 μg/ml of peptide ACYEFLWGPRALVETS, washed, and incubated(20,000 cells) with 5,000 cells of clones 1, 2 or 3. IFN-γ productionwas measured by ELISA after overnight co-culture. The results shownrepresent the average of triplicate co-cultures.

The binding of peptide MAGE-3₂₆₇₋₂₈₉ to purified HLA-DR molecules ofvarious allotypes was tested in competition assays using HLA-DR specificbiotinylated peptides (Table 4). The MAGE-3 peptide displayed a veryhigh affinity for DR1, binding more efficiently than the referencepeptide (Roche and Cresswell, J. Immunol. 144:1849, 1990). It also boundto the following molecules, listed by decreasing affinity: DR11, DRB5,DR15, DR4, DRB4 and DR7. No binding was observed on DR3, DR13 and DRB3.

TABLE 4 Binding of peptide MAGE-3₂₆₇₋₂₈₂ to multiple HLA-DRmolecules^(a) HLA-DR IC₅₀ (nM) of IC₅₀ (nM) of Ratios IC₅₀ MAGE- allelesreference peptides^(b) MAGE-3₂₆₇₋₂₈₂ 3/IC₅₀ reference DR1  2 (±1)  0.4(±0.2) 0.2 DR3 350 (±0) >10,000 — DR4  40 (±4)   730 (±4) 18 DR7  10(±3) 1,200 (±350) 120 DR11  25 (±0)   150 (±0) 6 DR13 390 (±140) >10,000— DR15  15 (±0)   220 (±35) 15 DRB3  20 (±0) >10,000 — DRB4  40 (±19)2,800 (±1,400) 70 DRB5  15 (±6)   170 (±140) 11 ^(a)The capacity of thepeptide MAGE-3₂₆₇₋₂₈₂ to bind multiple HLA-DR molecules was investigatedon purified DR molecules in competition assays using fluorescentreference peptides (± standard deviation). To facilitate the comparison,data are also presented as ratio between the IC₅₀ of the MAGE peptideand that of the reference peptide. A ratio lower than 10 indicatespeptides with a high affinity for a given HLA class II molecule, while aratio higher than 10 corresponds to intermediate binders. ^(b)Referencepeptides (unlabeled forms of peptides that bind DR molecules) aredescribed in Materials and Methods.Recognition of Tumor Cells

Proteins that carry an endosomal targeting sequence are directed to theendosomes, enabling the cell to present on class II molecules peptidesderived from internal proteins. For instance some melanocytic proteinsare specifically targeted to melanosomes, which are organelles derivedfrom the endocytic compartment (Wang et al., J. Immunol. 163:5820,1990). MAGE proteins do not contain signal sequences or endosomaltargeting sequences. Therefore, class II-restricted MAGE peptides arenot expected to be presented by the tumors expressing MAGE. In line withthis, we described two DR13-restricted MAGE-3 peptides and oneDR15-restricted MAGE-1 peptide that were not recognized by CD4⁺ T cellclones at the surface of tumor cells (Chaux et al., J. Exp. Med.189:767, 1999; Chaux et al., Eur. J. Immunol. 31:1910, 2001). However,we and others have identified MAGE-3 peptides presented by DR11 and DP4that were recognized on tumor cells expressing MAGE-3 (Schultz et al.,Cancer Res. 60:6272, 2000; Manici et al., J. Exp. Med. 189:871, 1999).How these peptides are processed and presented is unclear.

We tested the production of IFN-γ by the three CD4⁺ clones stimulated bythe melanoma lines NA41-MEL and MZ2-MEL.43, which express the DR1 andMAGE-3 genes. Clone 1 recognized the NA41-MEL cells, indicating thatMAGE-3 antigenic peptide can be naturally processed and presented bymelanoma cells (FIG. 7A). This CD4⁺ clone lysed also NA41-MEL cells andthe autologous EBV-B cells that were transduced with retro-Ii.MAGE3(FIG. 7B). The absence of recognition of MZ2-MEL.43 cannot be explainedby a lower level of expression of MAGE-3, as expression was equivalentin NA41-MEL and MZ2-MEL.43, as measured by semi-quantitative RT-PCR(data not shown). The two other CD4⁺ clones were not stimulated by themelanoma lines, although the avidity of the three clones was equivalent.

We compared the three CD4⁺ clones by testing their ability to bestimulated by dendritic cells loaded with decreasing concentrations ofprotein MAGE-3 and we observed that cells incubated with less than 10 nMof protein stimulated the three clones to produce 2 ng/ml of IFN-γ (FIG.8). Therefore, the direct recognition of tumors appears to be not onlydependent on the adequate expression of MAGE-3 or a high avidity of theCD4⁺ T cell clone.

Recognition of the Homologous MAGE Peptides

Peptide ACYEFLWGPRALVETS (SEQ ID NO:4) is encoded by MAGE-3 and MAGE-12genes. Homologous peptides encoded by other MAGE genes are only slightlydifferent from the MAGE-3/MAGE-12 peptide (FIG. 9A). All the peptidesshare the central amino acid sequence EFLWGPRA (SEQ ID NO:25). Thehomologous MAGE peptides were tested for their ability to stimulate theCD4 clones. The MAGE-2 (ACIEFLWGPRALIETS; SEQ ID NO:26) and MAGE-6(ACYEFLWGPRALIETS; SEQ ID NO:27) peptides were recognized by clones 2and 3 as efficiently as the MAGE-3 peptide, despite the replacement of atyrosine or a valine by isoleucines (FIG. 9A). Therefore a preferredpeptide has the amino acid sequence ACXEFLWGPRALXETS (SEQ ID NO:28). TheMAGE-11 peptide (ACYEFLWGPRAHAETS; SEQ ID NO:29) was recognized by clone3, despite the replacement of two amino acids. The MAGE-1 and MAGE-4peptides (each ARYEFLWGPRALAETS; SEQ ID NO:30) and MAGE-10 peptide(ARYEFLWGPRAHAEIR; SEQ ID NO:31) peptides were also recognized by clone3, when 20-times higher peptide concentrations were used. DR1 dendriticcells were incubated with lysates of EBV-B cells expressing MAGE-1,3, or4, and tested for their ability to stimulate clone 3 to produce IFN-γ(FIG. 9B). As expected, clone 3 was stimulated with cells pulsed withlysates of MAGE-3-expressing cells. Importantly, it was also stimulatedwith cells pulsed with lysates of MAGE-1 or 4-expressing cells,demonstrating that clone 3 is able to recognize naturally processedMAGE-1 or MAGE-4 proteins and not only synthetic peptides.

Based on the homologous MAGE sequences identified above, variantsequences are prepared by substitution of one or more amino acids(including additions or deletions) and tested for recognition by CTLclones, by recognition by cells transfected with the TCR clonesidentified herein, and/or by binding of tetramers of these same TCRs.The variant sequences that are tested include: AXXEFLWGPRAXXETS (SEQ IDNO:32), ACYEFLWGPRALVXTS (SEQ ID NO:33), ACXEFLWGPRALVXTS (SEQ IDNO:34), ACXEFLWGPRALXXTS (SEQ ID NO:35), AXXEFLWGPRALXXTS (SEQ IDNO:36), wherein the “X” amino acid is systematically varied to test eachpossible combination. Some of the variant peptides will have sequencesidentical to the MAGE peptide sequences identified above, and areincluded as positive controls in the assays.

In an additional series of experiments, variants of the peptidesGSDPACYEFLWGPRAL (SEQ ID NO:3) are prepared and tested. Based on theresults obtained above, the variant sequences that are tested include:GSDPACXEFLWGPRAL (SEQ ID NO:37), GSDPACYEFLWGPRAX (SEQ ID NO:38),GSDPACXEFLWGPRAX (SEQ ID NO:39), GSDPAXXEFLWGPRAL (SEQ ID NO:40),GSDPAXXEFLWGPRAX (SEQ ID NO:41) and XXXXACYEFLWGPRAL (SEQ ID NO:42),wherein the “X” amino acid is systematically varied to test eachpossible combination.

The peptide EFLWGPRAL (SEQ ID NO:63) is shared by the following MAGEproteins: MAGE-1, MAGE-2, MAGE-3, MAGE-4, MAGE-6, MAGE-8, MAGE-12, andMAGE-N, all of which are expressed in cancer cells. The shorter sequenceEFLWGPRA (SEQ ID NO:25) is found in additional tumor-associated MAGEproteins, including MAGE11, MAGE-B3, MAGE-C3, MAGE-B6 and the CTantigens CT7 (MAGE-C1) and CT10 (MAGE-C2, MAGE-E1). Thus peptidescontaining SEQ ID NO:25 (EFLWGPRA) or SEQ ID NO:63 are particularlyuseful as peptides that bind HLA class II molecules and that canstimulate an immune response against cells that express a number of MAGEproteins.

Other aspects of the invention will be clear to the skilled artisan andneed not be repeated here. Each reference cited herein is incorporatedby reference in its entirety.

The terms and expressions which have been employed are used as terms ofdescription and not of limitation, and there is no intention in the useof such terms and expressions of excluding any equivalents of thefeatures shown and described or portions thereof, it being recognizedthat various modifications are possible within the scope of theinvention.

1. An isolated MAGE HLA class II-binding peptide comprising SEQ IDNO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:31, SEQ IDNO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:38, SEQ IDNO:39, SEQ ID NO:40, SEQ ID NO:41, or SEQ ID NO:42 or consisting of SEQID NO:25, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:36, or SEQ ID NO:63,wherein the MAGE HLA class II-binding peptide does not include a fulllength MAGE protein.
 2. The isolated MAGE HLA class II-binding peptideof claim 1 wherein the isolated peptide comprises an amino acid sequenceselected from the group consisting of SEQ ID NO:26, SEQ ID NO:27, SEQ IDNO:28, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:34, SEQ IDNO:35, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ IDNO:41, and SEQ ID NO:42.
 3. The isolated MAGE HLA class II-bindingpeptide of claim 1 wherein the isolated peptide consists of an aminoacid sequence selected from the group consisting of SEQ ID NO:26, SEQ IDNO:27, SEQ ID NO:28 and SEQ ID NO:29.
 4. The isolated MAGE HLA classII-binding peptide of claim 3 wherein the isolated peptide consists ofthe amino acid sequence set forth as SEQ ID NO:26 or SEQ ID NO:27. 5.The isolated MAGE HLA class II-binding peptide of claim 1, wherein theisolated peptide comprises an endosomal targeting signal.
 6. Theisolated MAGE HLA class II-binding peptide of claim 5, wherein theendosomal targeting signal comprises an endosomal targeting portion ofhuman invariant chain Ii.
 7. The isolated MAGE HLA class II-bindingpeptide of claim 1 wherein the isolated peptide is non-hydrolyzable. 8.The isolated MAGE HLA class II-binding peptide of claim 7 wherein theisolated peptide is selected from the group consisting of peptidescomprising D-amino acids, peptides comprising a -psi[CH₂NH]-reducedamide peptide bond, peptides comprising a -psi[COCH₂]-ketomethylenepeptide bond, peptides comprising a -psi[CH(CN)NH]-(cyanomethylene)aminopeptide bond, peptides comprising a -psi[CH₂CH(OH)]-hydroxyethylenepeptide bond, peptides comprising a -psi[CH₂O]-peptide bond, andpeptides comprising a -psi[CH₂S]-thiomethylene peptide bond.
 9. Acomposition comprising one or more of the isolated MAGE HLA classII-binding peptides of claim 1 complexed with one or more isolated HLAclass II molecules.
 10. The composition of claim 9, wherein the numberof isolated MAGE HLA class II-binding peptides and the number ofisolated HLA class II molecules are equal.
 11. The composition of claim10, wherein the isolated MAGE HLA class II-binding peptides and theisolated MAGE HLA class II molecules are coupled as a tetramericmolecule of individual isolated MAGE HLA class II-binding peptides boundto individual isolated HLA class II molecules.
 12. The composition ofclaim 9, wherein the HLA class II molecules are DR1 molecules.
 13. Thecomposition of claim 9, wherein the MAGE HLA class II binding peptidesand the HLA class II molecules are covalently linked.
 14. Thecomposition of claim 13, wherein the covalent link between the MAGE HLAclass II binding peptides and the HLA class II molecules includes alinker molecule.
 15. A composition comprising an isolated HLA classI-binding peptide and an isolated MAGE HLA class II-binding peptide,wherein the isolated MAGE HLA class II-binding peptide comprises SEQ IDNO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:31, SEQ IDNO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:38, SEQ IDNO:39, SEQ ID NO:40, SEQ ID NO:41, or SEQ ID NO:42 or consisting of SEQID NO:25, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:36, or SEQ ID NO:63, andwherein the MAGE HLA class-II-binding peptide does not include a fulllength MAGE protein.
 16. The composition of claim 15, wherein the HLAclass I-binding peptide and the MAGE HLA class II-binding peptide arecombined as a polytope polypeptide.
 17. The composition of claim 15,wherein the isolated MAGE HLA class II-binding peptide comprises anamino acid sequence selected from the group consisting of SEQ ID NO:26,SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33,SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39,SEQ ID NO:40, SEQ ID NO:41, and SEQ ID NO:42.
 18. The composition ofclaim 15 , wherein the isolated MAGE HLA class II-binding peptideconsists of an amino acid sequence selected from the group consisting ofSEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28 and SEQ ID NO:29.
 19. Thecomposition of claim 18, wherein the isolated MAGE HLA class II-bindingpeptide consists of the amino acid sequence set forth as SEQ ID NO:26 orSEQ ID NO:27.
 20. The composition of claim 15, wherein the isolated MAGEHLA class II-binding peptide comprises an endosomal targeting signal.21. The composition of claim 20, wherein the endosomal targeting signalcomprises an endosomal targeting portion of human invariant chain Ii.